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TRANSFUSION MEDICINE
From the Beckman Institute for Advanced Science and
Technology and the Department of Biochemistry, University of Illinois,
Urbana, IL.
For many years, human hemoglobin (Hb) isolated from erythrocytes
has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with
various undesirable properties of cell-free Hb, including cost, overall
oxygen affinity, alterations in cooperativity, and ready dissociation
into toxic dimeric species. The use of total gene synthesis has
resulted in very high levels of functional human Hb expression in
Escherichia coli, but there remains a desire for effecting
the crosslinking of the hemoglobin tetramer and providing for ready
means for increasing the globular molecular weight. In this
communication, we report a novel method for linking alpha chains. By
circularly permuting one alpha sequence, the second alpha chain in the
Hb tetramer can be linked with glycine residues to form 2 bridges
across the central cavity. The second alpha chain thus presents its
amino and carboxyl termini on a solvent exposed surface, providing for
additional polymerization of oxygen-carrying subunits or attachment of
any other peptide-based therapeutic.
(Blood. 2002;100:299-305) A mechanistic and predictive understanding of how a
primary sequence can fold to a single tertiary structure remains
undefined. Since a thorough understanding of this process will enable
the rational design of unique protein structures, this remains an area
of active research. One theory suggests that short continuous regions
within a protein develop local interactions early in the folding
process to minimize the number of accessible structures and catalyze
the formation of a functional protein. Radical perturbations of protein
structure by circular permutation is one technique being used in an
attempt to explore this issue. In addition, utilization of circular
permutation itself can be exploited in the design of new protein structures.
A circularly permuted protein is created in 2 steps. First, the
original termini are linked to form a circular polypeptide. New termini
are then created by cleavage of a peptide bond at a location distant
from the original termini. Goldenberg and Creighton engineered the
first circularly permuted sequence by chemically condensing the termini
of bovine pancreatic trypsin inhibitor (BPTI) and generating new
termini by limited proteolysis.1 This circularly permuted
variant was shown to refold, in vitro, to a native functional
conformation. This seminal experiment suggested that the location of
the termini has little effect on the final 3-dimensional structure of
the protein. Luger and coworkers genetically circularly permuted
phosphoribosyl anthranilate isomerase.2 The
Escherichia coli translated variant was structurally and
functionally similar to the wild-type protein. Several other monomeric
proteins have since been circularly permuted through genetic
manipulations, all of which maintain a nativelike
function.3-11 Single subunits of multimeric proteins can
also withstand circularly permuted sequences. For example, circularly
permuted catalytic chains of aspartate transcarbamoylase combined in
vitro with regulatory chains produced a folded and functional
multimeric protein.12 However, all of these polypeptides
are single-domain cooperatively folding units.
More recently, circularly permuted variants of multidomain proteins
have been accomplished. Circular permutation within the NAD binding
domain of glyceraldehyde-3-phosphate dehydrogenase produced a
nativelike functional protein.13 The termini of the T4
lysozyme were also moved from one domain to another without significant
effects on the stability or function of the protein.14 These results suggest that local sequence continuity within defined structural domains is not required for proper protein folding.
The remarkable similarity between circularly permuted isomers suggests
that the linear organization of secondary structures has little effect
on the final 3-dimensional structures. In fact, Viguera et al have
addressed this question more directly by exhaustively permuting the SH3
domain of Human hemoglobin is a heterotetrameric protein which consists of 2 Furthermore, there is a significant amount of commercial interest in
stabilizing the hemoglobin tetramer with the introduction of covalent
linkages across the dimeric interface. Cell-free hemoglobin solutions
are being explored for use in surgical and traumatic blood loss.
However, hemoglobin dissociates into dimers within the vasculature and
the dimers are rapidly filtered by the kidneys. Numerous chemical
methods of crosslinking the dimers have been investigated. Only
one genetic fusion of globin subunits has been reported wherein 2 Genetic constructions
Several steps were required in the construction of the circularly
permuted hemoglobin (CpHb). Each vector utilized in the construction is
schematically represented in Figure 1.
First, a tandem fusion of 2
Expression operons were then constructed consisting of the Protein expression and purification
CpHb was purified by the following methods. Cell paste was allowed to
thaw over a stream of carbon monoxide (CO), and all buffers used during
the lysis and purification procedure were saturated with CO. Thawed
cells were resuspended in 5 times (wt/vol) 10 mM
NaH2PO4, pH 6.0, 1 mM
ethylenediaminetetraacetic acid (EDTA). Cells were lysed by 3 to 4 passes through a Stansted AO-116 cell disrupter (Stansted
Fluid, Stansted, United Kingdom). After cell lysis, 80 units/mL DNase
and 8 units/mL RNase were added to the mixture and allowed to incubate
at room temperature for 1 hour. The mixture was then centrifuged at
100 000g in a Beckman L8-M ultracentrifuge (Beckman
Instruments Incorporated, Fullerton, CA) for 30 minutes. The
supernatant was retained and the pH adjusted to 6.0 with 20 mM
NaH2PO4. The supernatant was then loaded onto a
carboxy methyl cellulose column (Whatman, Clifton, NJ)
equilibrated with 10 mM NaH2PO4, pH 6.0, 1 mM
EDTA. The column was then washed with 4 column volumes of 10 mM
NaH2PO4, pH 6.0, 1 mM EDTA, and finally eluted
with a step gradient to 20 mM Tris-HCl, pH 7.0, 1 mM EDTA. Further
purification was accomplished by high-pressure liquid chromatography.
The protein samples were first exchanged into Millipore water or 10 mM
NaH2PO4, pH 6.0, 1 mM EDTA using a Sephadex
G-25 column (Pharmacia Biotech, Peapack, NJ). The protein was
then loaded onto a HiLoad SP Sepharose column (Pharmacia
Biotech) equilibrated with 10 mM
NaH2PO4, pH 6.0, 1 mM EDTA. The column was
washed with one column volume of the equilibration buffer. The
circularly permuted hemoglobin was eluted with a linear gradient to 20 mM NaH2PO4, pH 7.0, 1 mM EDTA. The same method
was used for Di Protein characterization Electrospray mass spectometry was performed on the protein samples at the University of Illinois Mass Spectrometry Facility using a Quattro-70 mass spectrometer (Micromass, Manchester, United Kingdom). Purified protein samples were exchanged into water using a Sephadex G-25 column. The samples were diluted to a concentration of 10 pmol/µL into a 50:50 acetonitrile:water solution containing 0.2% formic acid for the experiments.N-terminal sequencing was performed at the University of Illinois Genetic Engineering Facility. One nanomole of protein was sequenced by automated edman degradation48 using a 470A gas phase sequencer (Applied Biosystems, Foster City, CA) with an online PTH amino-acid analyzer. Polyacrylamide gel electrophoresis (PAGE) was performed on the purified
protein samples. Sodium dodecyl sulfate (SDS)-PAGE was performed after
boiling in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2.5% SDS, 5%
Heme-globin stoichiometry To assess the heme-to-globin ratio in each construct, the protein content and heme content were measured independently. Protein samples were diluted to an approximate [heme] of 10 µM. Heme content was assessed by a pyridine hemochromogen assay.49 One mL of protein was added to 3 mL of 4.1 M pyridine, 0.1 M NaOH and allowed to incubate at room temperature for 1 hour. A few grains of dithionite were added to the sample; the sample was mixed and the absorbance at 556 nm was determined. The heme content was calculated using 556 = 0.032 µM 1cm1
for the reduced pyridine heme complex. The globin content was assessed
with the absorbance at 280 nm. The 280-nm absorbance is attributable to
aromatic side chains (phenylalanine, tyrosine, and tryptophan). Since
each globin domain contained an equal complement of these residues, the
absorbance at 280 is a good indicator of proportional globin content.
The ratios of these independent measures were normalized to a ratio of
4:1 for HbAo.
Spectroscopy methods Protein samples were reduced by the addition of a few grains of dithionite and exchanged into air-saturated water over a Sephadex G-25 column. The oxygen-bound spectrum was taken in a U-3300 spectrophotometer (Hitachi, Tokyo, Japan). Then a few grains of dithionite were added to the sample and a deoxy spectrum was taken in a septa-sealed cuvette. Finally, the CO bound form was generated by gently bubbling the sample with CO for 15 seconds.Circular dichroism spectra were recorded using a J-720
spectropolarimeter (Jasco, Victoria, British Columbia, Canada)
using a 0.1 cm pathlength quartz cell. The CD signal of the cuvette and
buffer was recorded and subtracted from the experimental samples. Protein solutions were adjusted to a heme concentration of 10 mM in 10 mM KPO4, pH 7.4, and CO saturated. Helical contents
were estimated using extinction coefficients at 222 nm as reported by
Greenfield and Fasman for Oxygen equilibrium Oxygen equilibrium analysis was performed using a modified Imai cell on an apparatus made in-house. The sample cell was described previously.44 White light from a tungsten source was chopped at a frequency of about 630 Hz by passing through an Oriel chopper (Oriel, Stratford, CT). Fiber bundles directed the light through the sample cell mounted over a homemade magnetic stirrer and into an Oriel 1/4 meter monochrometer where 560 nm or 430 nm light was isolated, depending on sample concentration. Signal detection was accomplished with a photomultiplier powered by a PS310 high-voltage power supply and amplified with a SR530 lock-in amplifier (all from Stanford Research Systems, Sunnyvale, CA). The cell was thermostated with a circulating water bath at 25°C. Gas exchange was accomplished by venting air or nitrogen over the sample surface after passing through a flow meter and a gas wash bottle thermostated in the circulating water bath. Gas flow was maintained at a rate to exchange ligands over a 45-minute period (about 20-30 mL/min). Oxygen concentrations were monitored with a YSI 5331 Clark electrode (Yellow Springs Instruments, Yellow Springs, OH). The polarization voltage of 0.8 volts for the electrode and current to voltage conversion were supplied by a Bioanalytic Systems CV-27 voltamograph (West Layfayette, IN). The voltage outputs for both the oxygen electrode and optical signals were recorded on Hewlett Packard 3840 multimeters (Hewlett-Packard, Palo Alto, CA), then transferred to a PC.The experiments were performed in 50 mM Tris-HCl, pH 7.4, 0.1 M
[Cl
Design of the circularly permuted hemoglobin Figure 3 is a schematic of the linear amino acid sequence and folded structure of the circularly permuted hemoglobin (CpHb). An open-reading frame coding for the circularly permuted Di globin was constructed. The coded polypeptide
consisted of the following: (1) an initiator methionine residue, (2)
residue 49-141 of an globin sequence (the first portion of a
circularly permuted globin), (3) a glycine residue, (4) a wild-type
globin sequence (the linking sequence), (5) a second glycine
residue, and (6) residue 1-48 of an globin sequence (the second
portion of a circularly permuted globin). The completed gene
construct was coexpressed with a wild-type  globin under the control
of a single promoter. As a control, the DiHb described by Looker et
al22 was also constructed. This protein consists of the
tandem fusion of 2 globins with a single linking glycine
residue.
Large structural transitions occur during the oxygenation of the
hemoglobin tetramer. High-resolution crystal structures of both the oxy
(R state) and deoxy (T state) states of hemoglobin were used in the
design of the circularly permuted Di The selection of a permutation site within the Biochemical characterization Both DiHb and CpHb were expressed to a high level in
E coli. The proteins were purified to homogeneity
and Edman degradation revealed that the amino terminal sequence of the
circularly permuted Di globin was consistent with cleavage of the
initiator methionine.29 This was expected from the rules
described previously for N-terminal processing in E
coli.30 Electrospray mass spectrometry, performed on
each variant, reconfirmed methionine cleavage of the CpHb globin
segments. Furthermore, each variant contained globin and globin
subunits of the expected molecular weight confirming that no
posttranslational modification of the subunits occurred in vivo (data
not reported).
SDS-PAGE also revealed that each variant consisted of 2 components,
The variant's capacity to bind heme was clear from the whole-cell CO
difference spectrum obtained for each protein. However, this did not
preclude the possibility that only a partial complement of heme was
binding to each protein. To assess the ratio of heme bound to globin
domains, the heme content and protein content were measured
independently as described in "Materials and methods." Each protein
was shown to contain a full complement of 4 hemes per tetrameric unit
(Table 1).
Spectroscopic characterization The electronic environments around the heme were investigated by the optical spectra of the oxygen-bound, carbon monoxide-bound, and deoxygenated species. The optical spectrum of each ligation state of each variant was nearly identical to HbA. The absorbance maximum for each variant and ligation state were all within 1 nm of HbA. Furthermore, the different ligation states were reversible since the carbon monoxide-bound state was competed off with oxygen and light, the deoxygenated state was created with the reduction of free oxygen by dithionite, and the carbon monoxide or oxygen-bound forms could be reformed with bubbling of the appropriate gas through a deoxygenated solution.The secondary structure of the proteins was assessed with far UV
circular dichroism spectroscopy. Helical contents were estimated to be
74%, 75%, and 78% for HbAo, Di Oxygen equilibrium Oxygen equilibrium measurements were taken to assess the ligand affinity and response to allosteric effectors. These measurements are exquisitely sensitive to the quaternary transitions and functional integrity of hemoglobin. Although DiHb exhibited an
oxygen affinity similar to HbA, CpHb displayed an oxygen
affinity that was 5-fold greater as evidenced by the partial pressure
(atmospheres) for 50% saturation (p50) of these proteins
listed in Table 2.
The cooperativity of the variants were described by the mathematical
model developed by Archibald Hill described previously.32 The Hill coefficients are reported as nmax in
Table 2. The ability of known allosteric regulators of hemoglobin (IHP
and protons) to shift the oxygen affinity was also investigated with
the circularly permuted variant. Both IHP and protons preferentially
bind to and stabilize the deoxy state of hemoglobin causing a right
shift in the oxygen-binding curve and increasing the overall
p50. The IHP effect is reported as the change in
p50 with the addition of 0.1 mM IHP, Table 2. The Bohr
effect, or increase in oxygen affinity with a decrease in pH was
determined by the independent measurement of oxygen affinity at 3 different pH values. The slope of this curve,
A variety of structural and functional techniques have been utilized in the investigation of the crosslinked and circularly permuted hemoglobin variants. Although there are minor differences, the recombinant proteins are remarkably similar to HbAo. The proteins assemble to the expected oligomeric structure and incorporate a full complement of heme. The identity of the optical spectra of 3 different ligation states suggests that the electronic environments of the heme cavities are similar. Furthermore, each protein folds to a structure with about 75% helical content consistent with HbAo. The only perturbations to these proteins were observed in the more sensitive functional assays. The decrease in oxygen-binding cooperativity is not surprising. The
Di Allosteric regulators of oxygen affinity play an important physiologic
role. These effectors bind to sites distinct from the heme cavity and
preferentially stabilize the deoxy state of hemoglobin. The ability of
the circularly permuted protein to respond to allosteric regulators
suggests that the protein is structurally similar to HbAo in these
regions distant from the heme cavity. IHP is a potent allosteric
regulator which binds with a significantly higher affinity to the same
cavity as 2,3 diphosphoglycerate.34-36 This cavity is
defined by several positively charged residues at the
Protons also preferentially bind to the T state of hemoglobin. In the
acidic environment of respiring tissues, the oxygen affinity of
hemoglobin is reduced to increase the release of oxygen. This results
from a change in acid-based equilibrium constant (pKa), between the T and R states, of several
titratable amino acids.37-39 Perutz et al38
proposed a mechanism for the Bohr effect in which the N-terminal amino
group of the Implications on protein folding and design The number of successfully engineered circular permutations and the identification of naturally occurring circular permutations is tribute to the remarkable insignificance of the termini location in the folding or stability of many proteins.43 This demonstrates that a vectorial folding mechanism, in which the protein folds in a linear fashion as it is translated, is not a requirement for a functional protein in vivo. However, it does not eliminate this as a possible folding pathway. It is likely that local regions of the protein fold to initiate more distant tertiary interactions. At least in some proteins the spatial uncoupling of protein regions will not prevent the protein from folding. In fact, peptide fragments have been shown to initiate protein folding independently then associate to form a complete protein.16-20 Work presented here extends this belief by demonstrating that the spatial separation of the circularly permuted globin fragments with a long linking domain (an globin)
will assemble to a functional protein.
The repeating nature of structural motifs in the CpDi Summary The successful construction of this multiglobin circular permutation opens up many possibilities in the future design of supramolecular protein structures, by suggesting that protein domains can be engineered within the circular permutation site of another protein. These new multidomain or multiprotein stuctures can be spatially oriented as desired by altering the circular permutation site of the primary protein, enabling the rational design of unique multidomain proteins. It could also enable the fixed approximation of proteins with physiologic protein-protein interactions as well as the pharmacuetical design of complexes that require distinct protein functionalities in close proximity.In the field of oxygen-carrying therapeutics this protein provides a
building block for the design of an enhanced oxygen delivery vehicle.
By linking the native The exposed termini of CpHb could also be utilized for the fusion of other proteins. Fusions with superoxide dismutase (SOD) (Sigma, St Louis), catalase, or other radical scavenging enzymes have the potential to minimize known toxic side effects of current oxygen-carrying therapeutics. Fusion with an erythropoeitin consensus sequence could be designed to induce endogenous red blood cell formation coincident with the in vivo degradation of the oxygen-carrying therapeutic. Alternatively, the defined vascular stability of human hemoglobin in plasma could be exploited as a pharmaceutically active peptide carrier for nearly any peptide-based product.
Thanks to Mark McLean and Aretta Weber for expert editorial assistance.
Submitted September 4, 2001; accepted February 13, 2002.
Supported by National Institutes of Health grants GM31756 and GM33775.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen G. Sligar, University of Illinois, 405 North Mathews, Urbana, IL 61801; e-mail: s-sligar{at}uiuc.edu.
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Top
Abstract
Introduction
-aminolevulinic
acid in 6 L shake flasks at 37°C. 
70°C.
log(p50)/