Lung surfactant protein A (SP-A) interactions with model lung surfactant lipids and an SP-B fragment.

Abstract

Surfactant protein A (SP-A) is the most abundant protein component of lung surfactant, a complex mixture of proteins and lipids. SP-A performs host defense activities and modulates the biophysical properties of surfactant in concerted action with surfactant protein B (SP-B). Current models of lung surfactant mechanism generally assume SP-A functions in its octadecameric form. However, one of the findings of this study is that when SP-A is bound to detergent and lipid micelles that mimic lung surfactant phospholipids, it exists predominantly as smaller oligomers, in sharp contrast to the much larger forms observed when alone in water. These investigations were carried out in sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG), and mixed LMPC + LMPG micelles, using solution and diffusion nuclear magnetic resonance (NMR) spectroscopy. We have also probed SP-A's interaction with Mini-B, a biologically active synthetic fragment of SP-B, in the presence of micelles. Despite variations in Mini-B's own interactions with micelles of different compositions, SP-A is found to interact with Mini-B in all micelle systems and perhaps to undergo a further structural rearrangement upon interacting with Mini-B. The degree of SP-A-Mini-B interaction appears to be dependent on the type of lipid headgroup and is likely mediated through the micelles, rather than direct binding.

Full text

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Published:May 09, 2011
r2011 American Chemical Society
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pubs.acs.org/biochemistry
Lung Surfactant Protein A (SP-A) Interactions with Model Lung
Surfactant Lipids and an SP-B Fragment
Muzaddid Sarker,†Donna Jackman,‡and Valerie Booth*,†,‡
†Department of Physics and Physical Oceanography and‡Department of Biochemistry, Memorial University of Newfoundland,
St. John’s, NL, Canada
b
S Supporting Information
L
air?water interface in alveoli and additionally provides the first
line of defense against inhaled microbes in the lungs. Surfactant
proteinA(SP-A)isthemostabundantproteincomponentofthe
lung surfactant system.1Substantial evidence indicates that SP-A
is a major contributor to innate host defense and inflammatory
immunomodulator processes of the lung.2?7SP-A may also play
aroleinthesurfaceactivityoflungsurfactant.Forinstance,SP-A
is essential for the formation of tubular myelin,8a potential
structuralprecursortothesurface-activefilm.SP-Aalsoenhances
adsorption of phospholipids along the air?water interface in
concerted action with surfactant protein B (SP-B)9,10and
induces calcium-dependent aggregation of lipid vesicles with or
without SP-B or surfactant protein C (SP-C).11,12Furthermore,
SP-Ahasbeenshowntoimprovethesurfaceactivityofsurfactant
under several challenging conditions such as the low surfactant
concentrations13andthepresenceofinhibitoryplasmaproteins14or
oxidants.15
SP-A is a multimeric glycoprotein. The capabilities of SP-A to
bind surfactant phospholipids, pathogen-associated molecular
patterns, and receptors on cell surfaces likely depend on its
complex oligomeric structure.16SP-A can assemble as a hexamer
of trimeric subunits; i.e., a total of 18 SP-A molecules may join
together to form the quaternary structure. This octadecameric
conformation is generally assumed to be the form in which the
protein carries out its biological functions.2,17
ung surfactant is a mixture of lipids and proteins that enables
normal breathing by reducing the surface tension at the
SP-A’s primary structure is highly conserved among different
mammalian species.18A single chain of human SP-A consists of
248 amino acids, as does the bovine SP-A used in this work.19Its
molecularweightvariesfromorganismtoorganism,from∼28to
36 kDa depending on the extent of post-translational modifica-
tions (e.g., glycosylation).20SP-A belongs to the structurally
homologousfamilyofinnateimmunedefenseproteinsknown as
collectins, so named for their collagen-like and lectin domains.17
It possesses four structural domains: a short N-terminal domain
that contains the cysteines required for intermolecular disulfide
bond formation, a proline-rich collagen-like domain that is
important for oligomerization, an R-helical coiled-coil neck
domain that is involved in trimerization, and a globular C-term-
inal carbohydrate recognition domain (CRD).2,17,21The high-
resolution crystal structures of recombinant trimeric CRD and
neckdomainsofratSP-A,inbothnativeandligand-boundforms,
have been determined (PDB IDs 1R13 and 1R14),22but the
complete structures of the full protein, its glycosylated form, or
higher oligomers are still unavailable.
SP-A is a hydrophilic and hence water-soluble protein. How-
ever, in the lungs, only about 10% of the total SP-A population is
found in the aqueous phase and almost 90% is lipid-associated,
the bulk of which is present within tubular myelin.23Therefore,
Received:
Revised:
February 2, 2011
April 27, 2011
ABSTRACT: Surfactant protein A (SP-A) is the most abundant
protein component of lung surfactant, a complex mixture of
proteins and lipids. SP-A performs host defense activities and
modulates the biophysical properties of surfactant in concerted
action with surfactant protein B (SP-B). Current models of lung
surfactant mechanism generally assume SP-A functions in its
octadecameric form. However, one of the findings of this study
is that when SP-A is bound to detergent and lipid micelles that
mimic lung surfactant phospholipids, it exists predominantly as
smaller oligomers, in sharp contrast to the much larger forms
observed when alone in water. These investigations were carried out in sodium dodecyl sulfate (SDS), dodecylphosphocholine
(DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG), and mixed LMPC þ LMPG
micelles, using solution and diffusion nuclear magnetic resonance (NMR) spectroscopy. We have also probed SP-A’s interaction
with Mini-B, a biologically active synthetic fragment of SP-B, in the presence of micelles. Despite variations in Mini-B’s own
interactions with micelles of different compositions, SP-A is found to interact with Mini-B in all micelle systems and perhaps to
undergo a further structural rearrangement upon interacting with Mini-B. The degree of SP-A?Mini-B interaction appears to be
dependent on the type of lipid headgroup and is likely mediated through the micelles, rather than direct binding.

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interactionswithphospholipidslikelyplayimportantrolesinSP-
A’s biological function. We have thus performed solution and
diffusion nuclear magnetic resonance (NMR) studies to inves-
tigatethelipidinteractionsandlevelofoligomerizationofbovine
SP-A, using an array of five different micelle systems mimicking
various lipid components of lung surfactant. The investigation
started with anionic sodium dodecyl sulfate (SDS) and zwitter-
ionic dodecylphosphocholine (DPC) micelles that are routinely
used in solution NMR studies of lipid-associated or membrane
proteins. It then proceeded to more physiologically relevant
micelle mimetics constituted from lysomyristoylphosphatidyl-
choline (LMPC), a single chain analogue of the surfactant phos-
pholipids containing the zwitterionic PC headgroup, and lyso-
myristoylphosphatidylglycerol (LMPG), a single chain analogue
of the surfactant phospholipids containing the anionic PG head-
group. Finally, a mixed LMPC (85%) þ LMPG (15%) micelle
systemwasusedapproximatingthephysiologicalratioofPCtoPG.
We have also used NMR techniques to probe the interaction
of SP-A with Mini-B in all these micelle systems. Mini-B is a
synthetic construct comprising the N- and C-terminal helices of
SP-B. Measurements of blood oxygenation and dynamic lung
compliance of surfactant deficient rat models show that Mini-B
performs as well as the full-length protein.24Thus, Mini-B likely
encompasses the key functional regions of SP-B. SP-B itself is
essential for lung surfactant function.25,26There are several indi-
cationsofinteraction,eitherdirectorindirect,betweenSP-Aand
SP-B. Although SP-A is not strictly required for the biophysical
function of lung surfactant,27it improves the surface activity of
lipid?protein preparations only if SP-B is present, especially in
the presence of anionic phospholipids.9,28The synergy between
SP-A and SP-B observed in the process of phospholipid mem-
brane fusion has been attributed to specific calcium-dependent
interactions between them.29,30Likewise, the perturbation of
dipalmitoylphosphatidylcholine(DPPC)/dipalmitoylphosphati-
dylglycerol (DPPG) bilayers by SP-A and SP-B together is
different from the sum of the effects of the individual proteins.31
The proteins also demonstrate a cooperative calcium-dependent
action in improving the resistance to surfactant inhibition by
blood and plasma proteins.32However, the most dramatic
exhibition of a concerted action is probably the in vitro recon-
stitution of tubular myelin when SP-A and SP-B are added to the
mixtures of DPPC and PG in the presence of calcium.33?35
Knowledge of the high-resolution structure of Mini-B,36along
withitsNMRchemicalshifts,providedanopportunitytodirectly
probeSP-A?Mini-Binteractionsinthepresenceofmodellipids.
SolutionNMRtechniquesarefrequentlyemployedinprobing
protein?protein interactions due to the sensitivity of NMR
chemical shift to the surrounding environment which allows
thebindingsurfaceofaproteintobemapped,merelybytitrating
in its binding partner and tracking the changes in the position
and/or intensity of the NMR signals.37However, there were
someadditionalcomplexitiesinvolvedinapplyingthisstrategyto
study the SP-A?Mini-B interaction. First, SP-A octadecamers
are about 504?648 kDa and thus very large for solution NMR
and are expected to give very broad, weak peaks in the spectra.
Second, since the hydrophobic Mini-B was solubilized in SDS
micellesforthestructuralstudies,itwasnecessarytocharacterize
Mini-B’s own interactions with various micelles in addition to
SP-A?micelle interactions before investigating any SP-A?Mini-B
interaction in the presence of those micelles.
’MATERIALS AND METHODS
Protein Preparation. SP-A was isolated and purified from
cow lungs, as described elsewhere.38,39The molecular mass of
SP-A (29.022 KDa) was confirmed by SDS?polyacrylamide gel
electrophoresis (SDS-PAGE) and matrix-assisted laser desorp-
tion/ionization?time-of-flight(MALDI-TOF)massspectrome-
try. Mini-B was produced by solid phase chemical synthesis
employing O-fluorenylmethyloxycarbonyl (Fmoc) chemistry
and purified by preparative reverse phase high performance
liquid chromatography (HPLC), as described elsewhere.36The
34-residue Mini-B possessed 9 backbone
acids: 6 leucines at positions 3, 7, 22, 25, 29, and 31, 2 alanines
at positions 6 and 13, and 1 glycine at position 18.
NMR Sample Preparation. SP-A samples were prepared in
aqueous solution (90% H2O þ 10% D2O) containing 0.4 mM
2,2-dimethyl-2-silapentane-5-sulfonate (DSS), 0.2 mM NaN3,
and 4.5 mM Hepes. SP-A?micelle samples were prepared by
addingthe required amounts of detergents/lipids tothe aqueous
sample. At least two samples were prepared for each micelle
system with differing ratios of the protein to detergent/lipid.
However, for each sample, the molar concentration of the deter-
gent/lipidwaskeptatleast200timeshigherthanthemonomeric
concentration of SP-A. The exact protein and detergent/lipid
concentrations of the samples are mentioned in the captions of
Figures 1?4. For SDS and DPC samples, deuterated (98%)
detergents, purchased from Cambridge Isotope Laboratories
(Andover, MA), were used. For LMPC and LMPG samples,
nondeuterated lipids, purchased from Avanti Polar Lipids
(Alabaster, AL), were used as their deuterated versions were
notcommerciallyavailable.ThesamplesweresettopH6.9using
NaOH and HCl solutions. Mini-B samples were prepared
separately, maintainingidenticalconditions tothe SP-A samples.
Finally, SP-A and Mini-B samples in each micelle system were
mixed together at equal quantities (i.e., a protein monomer ratio
of 1 to 1) to prepare the mixed protein samples.
15N-labeled amino
Figure 1. HN regions of 1D1H NMR spectra of SP-A in water and in
differentmicelleenvironments.(A)0.2mMSP-Ainwaterandin40mM
SDSand40mMDPC(256scans).(B)0.25mMSP-Ain50mMLMPC,
50 mMLMPG, and42.5 mM LMPCþ 7.5mM LMPG(160 scans). All
spectra within each panel are shown with the same intensity scale.
However, the intensity scales are not comparable between the panels as
sample compositions and acquisition parameters were different.

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Collection and Processing of Solution NMR Data. NMR
spectra were acquired on a Bruker Avance II 14.1 T (600 MHz)
spectrometer (Billerica, MA) using an inverse triple resonance
TXI probe. Data were collected and processed using Bruker
Topspin2.0.1D1HexperimentswereperformedforSP-Ainwater,
SP-A?micelle, Mini-B?micelle, and SP-A?Mini-B?micelle sys-
tems. 2D15N?1H HSQC experiments were performed for the
samples containing Mini-B. All experiments were performed at
37 ?C to match the physiological temperature. 1D1H spectra
were acquired with 128?320 scans using the water-gate water
suppression technique40and processed using an exponential
apodization function with 1 Hz line broadening. 2D15N?1H
HSQC spectra were acquired with 160?320 scans using the
flip-back water suppression technique41and processed using the
Qsine apodization function with a sine bell shift of 2. Although
theNMRexperimentswereperformedforatleasttwoseparately
prepared samples of each system, spectra of both samples
essentially looked identical.
Collection and Processing of Diffusion NMR Data. Diffu-
sion-ordered spectroscopy (DOSY) experiments were perfor-
med on the same Bruker Avance II 14.1 T (600 MHz) spectro-
meteremployingpulsedfieldgradient(PFG)NMR.42Thepulse
sequenceusedastimulatedechowithbipolargradientpulsesand
one spoil gradient,43followed by a 3?9?19 pulse for water
suppression.44The1H signals were attenuated to ∼5% of their
initial amplitudes by increasing the gradient strength from ∼2%
to95%in32steps.Experimentswereperformedat37?CforSP-
A in water and SDS and DPC samples, but at 25 ?C for LMPC,
Figure 2. Translational diffusion measurements of SP-A in water, SDS, and DPC micelles. Top panels show the 2D DOSY spectra of 0.2 mM SP-A in
water(A),0.2mMSP-Ain40mMSDS(B),and0.2mMSP-Ain40mMDPC(C).Bottompanelsshowthelinearfitsobtainedfortheattenuationofthe
integrated HN region of SP-A in water (D), in complex with SDS (E), and in complex with DPC (F). The linear fits for pure SDS (40 mM) and DPC
(40 mM) micelles, obtained from the attenuation of the peak at 0.80 ppm, are included in (E) and (F) for comparison.
Figure3. Translational diffusionmeasurements ofSP-AandMini-BinLMPC, LMPG,andLMPC(85%)þLMPG(15%)micellesystems. 2DDOSY
data were acquired separately for pure micelles (50 mM), SP-A (0.25 mM) in micelles (50 mM), Mini-B (0.25 mM) in micelles (50 mM), and SP-A
(0.125mM)þMini-B(0.125mM)inmicelles(50mM).Linearfitsshowtheattenuationofthe1Hsignalsformicellesandprotein?micellecomplexes
as determined from the lipid peak at 0.86 ppm.

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LMPG, and LMPC þ LMPG samples to minimize the effect of
thermal convection. The pseudo 2D DOSY spectra were pro-
ducedusingBrukerTopspin2.0.Theintegratedsignalintensities
were exported to Igor Pro for curve fitting. The translational
diffusion coefficient, DC, was derived from the 1D1H experi-
ments underlying the 2D DOSY, using the equation for the
attenuation of signal
ln½SðkÞ=Sð0Þ? ¼ ?DCk
with
k ¼ γ2g2δ2ðΔ ? δ=3Þ
where S(k) is the observed signal intensity, S(0) is the unatte-
nuated signal intensity, γ is the gyromagnetic ratio of the
observed nucleus (1H), g is the gradient strength (maximum
amplitude 35 G/cm), δ is the gradient pulse length (optimized
between 3 and 8 ms), and Δ is the diffusion time (100 ms). The
diffusion coefficient was determined from the slope of the linear
fitforln[S(k)/S(0)]versusk.Theobserveddiffusioncoefficient,
DC, was converted into apparent hydrodynamic diameter, dHA,
using the Stokes?Einstein equation
DC¼ kBT=3πηdHA
where kBis the Boltzmann constant, T is the absolute tempera-
ture, andηistheviscosityofthesolution (8.91?10?4kg/(m s)
at 25 ?C or 6.92 ? 10?4kg/(m s) at 37 ?C).
For each system, translational diffusion measurements were
performedusingmultiplepeaks,andtheaveragevalueofDC,and
corresponding dHA, was determined. In deuterated SDS and
DPC micelles, two separate values of average DCand dHAwere
calculated from the attenuation of detergent peaks at 0.80 and
1.22 ppm and protein peaks at 0.92 ppm and the integrated HN
region. However, in nondeuterated LMPC, LMPG, and LMPC þ
LMPG micelles, the signals from the lipids overwhelmed the
signalsfromtheprotein,andso,theaverageDCanddHAforthese
systems were calculated from the lipid peaks only. Four LMPC/
LMPGpeaksat0.86,1.28,1.59,and2.37ppmwereused.ForSP-A
in water, the average DCand dHAwere calculated using the peak
at 2.03 ppm and the integrated HN region.
For SDS and DPC samples, because the diffusion measure-
ments were obtained from the protein peaks in addition to the
detergent peaks, the subpopulations of protein?micelle com-
plexes, Scomplex, and protein?free micelles, Smicelle(= 1 ? Scomplex)
(Supporting Information, Table S1), were determined using a
two-site model45
DCðobservedÞ¼ ScomplexDCðcomplexÞþ ð1 ? ScomplexÞDCðmicelleÞ
where DC(observed)is the observed diffusion coefficient of the
protein?micelle sample and DC(complex)and DC(micelle)are the
diffusioncoefficientsoftheprotein?micellecomplexesandpure
micelles, respectively.
’RESULTS
SP-A?Micelle Interactions. 1D1H spectra of SP-A in water
and in different micelle environments were acquired to obtain
indications of the protein conformation. Figure 1 shows the HN
regions (6.2?8.7 ppm) of 1D1Hspectra of SP-A in water and in
different micelle environments. In water, very few signals are
observed, and those are broad and weak, as expected for a high
molecular mass protein. The few observable signals are likely
generated by some highly mobile region(s) of SP-A undergoing
fastmotion(e.g.,aflexibleloop).Interestingly,drasticchangesin
the SP-A spectra are observed with the addition of detergent or
Figure4. 2D15N?1HHSQCspectraofMini-Bindifferentmicellesintheabsence(toppanels)andpresence(bottompanels)ofSP-A.0.2mMMini-B
(A) and 0.1 mM Mini-B þ 0.1 mM SP-A (F) in 40 mM SDS. 0.2 mM Mini-B (B) and 0.1 mM Mini-B þ 0.1 mM SP-A (G) in 40 mM DPC. 0.25 mM
Mini-B (C) and 0.125 mM Mini-B þ 0.125 mM SP-A (H) in 50 mM LMPC. 0.25 mM Mini-B (D) and 0.125 mM Mini-B þ 0.125 mM SP-A (I) in
50 mM LMPG. 0.25 mM Mini-B (E) and 0.125 mM Mini-B þ 0.125 mM SP-A (J) in 42.5 mM LMPC þ 7.5 mM LMPG. Spectra A?E were acquired
using 160 scans, and spectra F?J were acquired using 320 scans.

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lipid micelles. TheHNregionsdisplaymanyintenseanddispersed
signals consistent with a substantially lower SP-A molecular mass
than observed in the absence of micelles.
To address this apparently substantial change of the SP-A
oligomerizationstateuponadditionofmicelles,2DDOSYexper-
iments were performed to estimate the size of the complexes
(Figures2and3,Table1).ForSP-Aaloneinwater,theapparent
hydrodynamic diameter, dHA, is 11.11 ( 1.48 nm. By contrast,
thedHAofSP-A?micellecomplexesaremuchsmallerinSDSand
DPC.ForSP-A?SDS,thedHAare3.27(0.93and6.30(0.94nm,
as measured using the SDS peaks and SP-A peaks, respectively.
Similarly,forSP-A?DPC,thedHAmeasuredfromtheDPCpeaks
and SP-A peaks are 3.57 ( 0.21 and 4.20 ( 0.21 nm, respectively.
ThedHAofpuremicelleswerealsomeasuredforcomparisonand
found to be 1.22 ( 0.02 nm for SDS and 1.96 ( 0.01 for DPC,
which conform well to what has been found by others for low
SDS concentrations.46It is normal to obtain different diffusion
coefficients from the detergent or lipid peaks compared to the
protein peaks of a protein?micelle sample, since the observed
value is the weighted average of the free and bound species.47
And, this allows for the calculation of the relative populations of
free and protein-bound micelles. On the basisoftheapplicationof
atwo-sitemodel(SupportingInformation, TableS1),45it isfound
that 76% of the SDS micelles and 85% of the DPC micelles are
bound to SP-A, while the rest remain as protein-free micelles.
Translational diffusion measurements of the more physiolo-
gically relevant micelle systems indicate dHAof 7.30 ( 0.10,
8.31 ( 0.03, and 8.40 ( 0.13 nm for LMPC, LMPG, and
LMPCþLMPG micelles, respectively. When these micelles
are bound to SP-A, the dHAof the complexes are increased to
10.37 ( 0.21, 11.27 ( 0.08, and 10.83 ( 0.22 nm, respectively,
as measured using the same lipid peaks. These dHAare still sub-
stantially smaller than what would be expected for an octadeca-
meric SP-A?micellecomplex(SupportingInformation,TableS2).
The effects of the interaction with micelles on the oligomeric
stateofSP-Awerealsosupportedbytheresultsofanonreducing
SDS-PAGE (data not shown). No band was visible for SP-A in
water, indicating a protein mass too large to enter the separating
gel (i.e., >100 kDa). In DPC, a band at ∼60 kDa was seen,
correspondingtothemass ofanSP-Adimer.AndinSDS,aband
at ∼28 kDa was seen, corresponding to the mass of an SP-A
monomer.
Mini-B?Micelle Interactions. Unlike SP-A, Mini-B was not
soluble in water, and hence no experiments with Mini-B in the
absence of micelles were possible. However, like SP-A, Mini-B
also modifies the diffusion coefficients of all the micelle types
Table 1. Average Observed Translational Diffusion Coefficients and Corresponding Apparent Hydrodynamic Diameters of
Detergent/Lipid Micelles and Protein?Micelle Complexes As Calculated from the DOSY Signal Attenuation
compositionpeaks fromobserved diffusion coefficient ?10?10(m2/s)
SP-A in waterSP-A
SDS micellesSDS
SP-A?SDS complex
SP-A
Mini-B?SDS complex
Mini-B
SP-A?Mini-B?SDS complex: fit 1
protein
SP-A?Mini-B?SDS complex: fit 2
protein
DPC micellesDPC
SP-A?DPC complex
SP-A
Mini-B?DPC complex
Mini-B
SP-A?Mini-B?DPC complex: fit 1
protein
SP-A?Mini-B?DPC complex: fit 2
protein
LMPC micelles LMPC
SP-A?LMPC complex
Mini-B?LMPC complex
SP-A?Mini-B?LMPC complex
LMPG micelles LMPG
SP-A?LMPG complex
Mini-B?LMPG complex
SP-A?Mini-B?LMPG complex
LMPCþLMPG micelles
SP-A?LMPCþLMPG complex
Mini-B?LMPCþLMPG complex
SP-A?Mini-B?LMPCþLMPG complex
apparent hydrodynamic diameter (nm)
0.596 ( 0.079
5.395 ( 0.101
2.091 ( 0.595
1.055 ( 0.158
3.460 ( 0.052
2.696 ( 0.267
1.661 ( 0.072
0.993 ( 0.041
0.501 ( 0.028
0.328 ( 0.014
3.362 ( 0.008
1.837 ( 0.110
1.566 ( 0.077
2.621 ( 0.051
2.591 ( 0.359
1.068 ( 0.206
0.561 ( 0.070
0.549 ( 0.037
0.327 ( 0.007
0.671 ( 0.009
0.472 ( 0.009
0.737 ( 0.002
0.572 ( 0.008
0.589 ( 0.002
0.435 ( 0.003
0.682 ( 0.005
0.537 ( 0.007
0.583 ( 0.009
0.452 ( 0.009
0.745 ( 0.022
0.538 ( 0.009
11.11 ( 1.48
1.22 ( 0.02
3.27 ( 0.93
6.30 ( 0.94
1.90 ( 0.03
2.45 ( 0.25
3.95 ( 0.17
6.61 ( 0.27
13.12 ( 0.74
20.02 ( 0.86
1.96 ( 0.01
3.57 ( 0.21
4.20 ( 0.21
2.51 ( 0.04
2.56 ( 0.35
6.26 ( 1.21
11.80 ( 1.48
11.99 ( 082
20.07 ( 0.43
7.30 ( 0.10
10.37 ( 0.21
6.65 ( 0.02
8.56 ( 0.11
8.31 ( 0.03
11.27 ( 0.08
7.18 ( 0.05
9.13 ( 0.12
8.40 ( 0.13
10.83 ( 0.22
6.57 ( 0.20
9.10 ( 0.15
SDS
SDS
SDS
SDS
DPC
DPC
DPC
DPC
LMPC
LMPC
LMPC
LMPG
LMPG
LMPG
LMPCþLMPG
LMPCþLMPG
LMPCþLMPG
LMPCþLMPG

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(Table 1), although, interesting differences are found between
how Mini-B interacts with SDS/DPC detergent micelles versus
LMPC/LMPG lipid micelles. The dHAof the complex, upon
inclusion of Mini-B, increases in detergent micelles but de-
creases in lipid micelles. While the dHAof the SDS micelle is
1.22 ( 0.02 nm, that for the Mini-B?SDS complex increases to
1.90 ( 0.03 and 2.45 ( 0.25 nm when calculated from the SDS
and Mini-B peaks, respectively. Similarly, the dHAof the DPC
micelle is 1.96 ( 0.01 nm, but this increases to 2.51 ( 0.04 and
2.56 ( 0.35 nm for the Mini-B?DPC complex when calculated
from the DPC and Mini-B peaks, respectively. On the other
hand, the dHAof LMPC, LMPG, and LMPCþLMPG micelles
are 7.30 ( 0.10, 8.31 ( 0.03, and 8.40 ( 0.13 nm, respectively,
but upon inclusion of Mini-B, the dHAof the peptide?micelle
complexesdecreaseto6.65(0.02,7.18(0.05,and6.57(0.20nm,
respectively.Estimationofsubpopulations,basedonthetwo-site
model(SupportingInformation,TableS1),indicatesthat72%of
the SDS micelles and 96% of the DPC micelles are in complex
with Mini-B, while the remainder exist as protein-free micelles.
1D
acquired to obtain indications of peptide conformation in the
micelle systems. The 6?9 ppm regions of 1D1H spectra of
Mini-B display well-dispersed HN signals for all micelle compo-
sitions (not shown). However, 2D15N?1H HSQC spectra in-
dicate differences in Mini-B’s conformation in anionic versus
zwitterionic micelles as well as detergent versus lipid micelles
(Figure 4A?E). All nine HSQC peaks are seen in SDS and
LMPG micelles, but the peak for Gly18 (assigned in ref 36)
is missing in DPC and LMPC micelles. Interestingly, in
LMPCþLMPG micelles, the Gly18 peak is present, although
the mixed micelles contain only 15% LMPG. There are several
additional weak peaks present for Mini-B in LMPC, LMPG, and
LMPCþLMPG but only a few in SDS and DPC, indicating
1H and 2D
15N?1H HSQC spectra of Mini-B were
greater conformational heterogeneity in the lipid versus deter-
gent micelles.
SP-A?Mini-B Interactions. Experiments to probe any inter-
actionbetweenSP-AandMini-Binthepresenceofmicelleswere
performed using mixtures containing equimolar monomeric
concentrations of each protein. 1D1H spectra of Mini-B?SP-A
mixtures(notshown)lookalmostidenticaltothatofSP-Aalone.
This is not unexpected as SP-A has more than 7 times as many
amino acids as Mini-B.
Figure4F?Jdisplaysthe15N?1HHSQCspectraofMini-Bafter
the additionof SP-A. Inanionic and mixed micelles,allninemini-B
peaks, and the additional weaker peaks, remain unaffected by the
inclusionofSP-A.Inzwitterionicmicelles,however,almostallMini-
B peaks disappear when SP-A is present, leaving very weak traces
of only a few. This likely indicates that all or most of Mini-B
are bound in complexes, presumably complexes of SP-A?micelle,
whicharetoolargetoyieldtheHSQCsignals.Sincethereisenough
detergent/lipid present to provide more than twice as many zwit-
terionic micelles as Mini-B molecules, it seems that Mini-B has a
strong preference to interact with SP-A?micelle complexes over
micelles without SP-A. This interpretation is further supported by
the absence of any changes to the missing or weak HSQC peaks of
Mini-B even when extra DPC is added (not shown).
Since 2D HSQC spectra suggested that, upon addition of
SP-A, there was likely a substantial increase in the size of Mini-B
complexes in zwitterionic micelles but no major change in
anionic or mixed micelles, we performed translational diffusion
measurements to probe the change in size for all systems.
Interestingly, 2D DOSY spectra of the SP-A?Mini-B mixture,
when compared to that of the individual proteins, demonstrate a
changeindHAforallmicellecompositions.AsshowninFigure5,
the signal attenuation curves for SP-A?Mini-B mixtures in SDS
and DPC micelles do not fit well with a single line (i.e., a single
component fit). However, approximately the first and the last
halves of the data are fit well with two lines having two different
slopes [i.e., a two-component fit48]. Thus, two diffusion coeffi-
cients are obtained, and there are, at least, two distinct sub-
populationsofprotein?micellecomplexespresentinthesample.
The diffusion coefficients and corresponding hydrodynamic
diameters measured from the two fits are reported in Table 1.
In SDS, the dHAof the SP-A?Mini-B subpopulations are
6.61 ( 0.27 and 20.02 ( 0.86 nm, as measured from the protein
peaks. Although the dHAof the first subpopulation is not signifi-
cantly different from the SP-A?SDS complex (6.30 ( 0.94 nm),
that of the second subpopulation is much larger. Hence, a
fraction of the total Mini-B and SP-A molecules present in the
mixture likely form large combined protein?micelle complexes.
The approximate ratio of the small-to-large subpopulations of
Mini-B?SP-A?SDS is 85%:15%, as estimated from the y-axis
(relative signal intensity) intercepts of the two linear fits for the
HNsignalattenuation.InDPC,ontheotherhand,thedHAofthe
SP-A?Mini-Bsubpopulationsare11.80(1.48and20.07(0.43nm
as measured from the protein peaks. In this case, the dHAof
both subpopulations are much larger than that of SP-A?DPC
(4.20(0.21nm)orMini-B?DPC(2.56(0.35nm)complexes.
The approximate ratio of small-to-large subpopulations of Mini-
B?SP-A?DPC is 62%:38%, as estimated from the y-axis inter-
ceptsofthetwolinearfitsfortheHNsignalattenuation.Thus,in
DPC,perhapstheentirepopulationsofSP-AandMini-Binteract
to form larger complexes, but with heterogeneous sizes.
Interestingly, the translational diffusion measurements in
LMPC, LMPG, and LMPCþLMPG micelles demonstrate quite
Figure 5. Signal attenuation curves obtained from the translational
diffusion measurements of 0.1 mM SP-A þ 0.1 mM Mini-B in 40 mM
SDS (A) and in 40 mM DPC (B). None of the curves fit well with a
single line. However, approximately the first and the last halves of the
data fit well with two lines having two different slopes. Consequently,
two diffusion coefficients are obtained for each system.

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different results from SDS and DPC micelles (Table 1). First,
each of the signal attenuation curves for SP-A?Mini-B?micelle
constitutes a single linear fit and hence yields a single diffusion
coefficient. Thus, complexes with only a single homogeneous
hydrodynamic diameter are apparently present for SP-A?Mini-
B in LMPC, LMPG, and LMPCþLMPG systems. Second, the
dHAof SP-A?Mini-B?micelle complexes are larger than Mini-
B?micelle complexes but, surprisingly, smaller than SP-A?
micelle complexes. The dHAof SP-A?Mini-B?LMPC is 8.56 (
0.11 nm as opposed to 6.65 ( 0.02 nm for Mini-B?LMPC and
10.37 ( 0.21 nm for SP-A?LMPC. Similarly, the dHA of
SP-A?Mini-B?LMPG is 9.13 ( 0.12 nm, but that of Mini-
B?LMPGis7.18(0.05nmandSP-A?LMPGis11.27(0.08nm.
Also in mixed micelles, the dHA are 9.10 ( 0.15 nm for
SP-A?Mini-B?LMPCþLMPG, 6.57 ( 0.20 for Mini-B?
LMPCþLMPG,and10.83(0.22 nm forSP-A?LMPCþLMPG.
To check if the observed micelle-bound SP-A represents most
oftheproteinpopulationoriftheremightbeasignificantfraction
of the SP-A molecules not giving rise to observable signals, we
performed a comparison between SP-A and Mini-B signal
intensities in all micelle systems (Supporting Information, Table S3).
When the tallest peaks in the HN regions, normalized with
respect to DSS peak intensity, are compared, SP-A exhibits
higher signal intensity than Mini-B by 22 times in SDS, 8 times
in DPC, 6 times in LMPC, 5 times in LMPG, and 4 times in
LMPCþLMPG. Thus, since all or most of the micelle-bound
Mini-B is likely visible in the NMR spectra, most of the SP-A in
the micelle samples is also likely being observed, at least in the
absence of Mini-B. On the other hand, mixed SP-A?Mini-B
exhibits8,4,4,2,and2timeshighersignalintensity,respectively,
in the five micelle systems, when compared to Mini-B. Thus, in
the mixed protein samples, significant fractions of the total
populations appear to be absent from the spectra, presumably
because their complexes are too large to observe by solution
NMR. These large complexes are likely formed by interaction
between SP-A and Mini-B.
’DISCUSSION
Our initial NMR studies aimed at characterizing SP-A?lipid
and SP-A?Mini-B interactions gave rise to a surprising result.
While 1D1H NMR spectra of SP-A in water displayed the broad
and weak peaks expected for a protein the size of an SP-A
octadecamer, when micelles were added, the spectra of SP-A
changed completely (Figure 1). In the presence of micelles, the
spectra of SP-A exhibited relatively intense, resolved, and dis-
persed peaks, typical of a much smaller protein than an SP-A
octadecamer. To estimate the size of the protein complex
consistent with these NMR spectra, we calculated the expected
line width and intensity (which depend on the rotational
correlation time and hence size) for different oligomeric forms
of SP-A (Supporting Information, Table S4). For example, the
expected line width for an SP-A monomer would be ∼11 Hz as
opposedto∼147Hzforanoctadecamer,andthesignalintensity
of a monomer would be ∼14 times greater than that for an
octadecamer. Thus, the appearance of the SP-A spectra in SDS
andDPCmicellesareconsistent withacomplexinthesizerange
of an SP-A monomer to trimer. Also, even though the spectra of
SP-A in LMPC, LMPG, and LMPCþLMPG micelles appear to
originate from a somewhat larger complex compared to the
spectraofSP-AinSDSandDPCmicelles,theyarestillconsistent
withamicelle-boundSP-Asubstantiallysmallerthananoctadecamer.
To quantify the oligomeric states of the micelle-bound SP-A,
we employed DOSY NMR techniques to obtain translational
diffusion measurements. DOSY experiments can reflect a variety
of parameters, including the fractions of free and bound species,
crowding, shape, and, most prominently, the size. In general, the
observed single component diffusion coefficient of a micelle
sample corresponds to the weighted average of free and bound
species when the rate of exchange is fast on the NMR time
scale.45Separate diffusion measurements from SDS/DPC and
protein peaks for theprotein?micelle samples (facilitated bythe
use of deuterated detergents) allowed us to calculate the fraction
of micelles forming complexes with the proteins. In terms of the
potential crowding effects, although the particles can experience
obstructed diffusion at high concentrations (e.g., g100 mM
SDS),49thesewerenotexpectedtoaffectthedataattherelatively
low concentrations employed for this study (e50 mM deter-
gent/lipid). While the particle shape indeed affects translational
diffusion, the effects of shape changes are small compared to
changes in size. For example, ellipsoidal particles 5 times as long
as they are wide diffuse only 25% more slowly than spherical
particles of the same size.50Furthermore, unlike rotational
diffusion measurements, translational diffusion measurements
are not affected by changes in protein flexibility.50We have thus
chosen to focus our interpretation of the DOSY data largely on
the complex size. For this reason, and because the particle
diameter is more intuitive to grasp than the diffusion coefficient,
we have converted the diffusion coefficient (DC) to the apparent
hydrodynamic diameter (dHA), i.e., the diameter of a sphere
apparently diffusing at the same rate, using the Stokes?Einstein
equation.
Diffusion measurements demonstrate that dHAfor micelles of
all compositions increase substantially upon addition of SP-A,
reflecting the formation of detergent/lipid?protein complexes
(Table 1 and Figure 6). An analysis based on a two-site model
(SupportingInformation,TableS1)45indicatesthat,forSDSand
DPC systems, more than three-fourths of the micelles are
involved in the formation of complexes with SP-A. Interestingly,
the dHAfor SP-A?SDS and SP-A?DPC complexes are more
than 3 times smaller than that for SP-A alone in the absence of
micelles. However, for SP-A in complex with LMPC, LMPG, or
LMPCþLMPG micelles, the dHAare similar to that of SP-A
alone in water. To interpret what this means in terms of the
oligomeric state of SP-A within various micelles, we have esti-
matedthe contributionofthe micelleitselftothediffusion ofthe
complex and used this to estimate the oligomeric state of the
SP-A within the micelle?SP-A complex. This analysis indicates
SP-A oligomeric states of approximately 10, 12, and 9 molecules
in LMPC, LMPG, and LMPCþLMPG micelles, respectively, as
well as oligomeric states of approximately 1 and 3 molecules in
DPC and SDS micelles, respectively (Supporting Information,
TableS5).Intheabsenceofmicelles,thediffusionmeasurements
indicate an oligomeric state of even larger than octadecamer
(18 molecules) for SP-A. Comparison of SP-A’s NMR signal
intensitywiththatofMini-B(SupportingInformation,TableS3)
indicates that the vast majority of SP-A molecules, if not the full
population, are observed in the NMR spectra acquired in the
presence of micelles; i.e., the observed signals are not generated
by just a small subpopulation of SP-A. There is, therefore, a
dramaticreductioninSP-A’soligomericstatewhentheproteinis
bound to micelles.
Presumably, not all of the monomers in the supramolecular
SP-Aassemblyarecovalentlyattachedbydisulfidebonds;rather,

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many of the subunits are associated only through noncovalent
interchain interactions. The addition of amphipathic lipids/
detergents appears to disrupt these noncovalent interactions
and thus cause the subunits to dissociate. It is plausible that
the electrostatic interactions, and perhaps the hydrophobic
interactions as well, between the protein and the lipid/detergent
molecules overwhelm many of SP-A’s intersubunit noncovalent
interactions,andthusmicellecomplexescontainingsmallerSP-A
oligomers are formed.
While micelles provide a surface of higher curvature when
compared to the planar surface of lipid bilayers, SP-A’s interac-
tions with curved surfaces are probably just as relevant as its
interactions with flat surfaces, given that many current models of
surfactant mechanisms show SP-A located at the highly curved
corners of tubular myelin.2Additionally, for membrane proteins
where crystal structures have been determined in complex with
lipids/detergents, these structures have been found to corre-
spond well with micelle-bound solution structures (e.g., ref 51).
It is thus likely, as in micelles, smaller oligomers of SP-A are also
present in native lipid environments.
FormationofsmalleroligomericformswhenSP-Aisboundto
micelles has consequences on our understanding of SP-A’s
functional mechanism, since SP-A’s biological roles, in relation
to either antimicrobial activities or surfactant biophysical activ-
ities,arealmostalwaysattributedtoitsoctadecamericstructure.2,17,19
This presumption derives from gel filtration and sedimentation
equilibriumstudies52aswellastransmissionelectronmicroscopy
(TEM)53performed with purified SP-A in lipid-free aqueous
solutions. However, gel filtration and sucrose density gradient
centrifugation of unpurified SP-A have indicated that the protein
does not exist purely as fully assembled octadecamers but is
consistently found insmaller oligomeric forms including a tetramer
of trimers (i.e., 12 molecules), dimer of trimers (i.e., 6 molecules),
dimer (i.e., 2 molecules), and even monomer (i.e., a single
molecule).54The TEM image of recombinant SP-A by itself also
displays smaller aggregates like tetramers, trimers and dimers, and
evenmonomersundermildreducingconditions.53TheTEMimage
oftubularmyelin,ontheotherhand,shows“X”-shapedstructuresin
thesquarelatticeregionswhicharemodeledasSP-Aoctadecamers.2,8
However, on the basis of the data present in this work, as well as
in studies such as in ref 54, it appears that it may be time to re-
examine the assumption that SP-A functions primarily as an
octadecamer.
These studies also reveal several aspects of Mini-B?lipid
interactions. First, as indicated by differences in the HSQC
spectra of Mini-B (Figure 4), the loop connecting Mini-B’s two
helices appears to take on a relatively stable conformation in
anionic and mixed micelles but undergoes conformational ex-
change at an intermediate rate in zwitterionic micelles. Second,
the DOSY data (Table 1 and Figure 6) indicate that while
complexes of Mini-B with SDS and DPC micelles are larger than
the micelles alone, the inclusion of Mini-B actually leads to a
decrease indHAof the micelles composedof LMPC, LMPG, and
LMPCþLMPG.ThemostlikelyexplanationforthisisthatMini-
B induces the formation of micelles with a smaller number of
lipids per micelle or causes the micelles to compactify. However,
itisalsopossiblethatMini-Bcausesthemicellestobecomemore
spherical. This ability of Mini-B to modulate highly curved lipid
structures is of importance in the consideration of the mechan-
ism of its parent protein, SP-B, which is frequently postulated to
act by promoting or modifying curved lipid structures.55,56
The differences observed between Mini-B’s effects on the small
detergentmicellesofSDSandDPCversusitseffectsonthelarger
lipid micelles of LMPC, LMPG, and LMPCþLMPG underline
thatthe protein?lipidinteractionsare governedbyfactorsmuch
more subtle than just the electrostatic charge of the headgroups.
The NMR data provide no indicationof any direct interaction
between SP-A and Mini-B, which would, for example, have been
supported by an SP-A-induced change in the chemical shifts of
Mini-B’s HSQC peaks (Figure 4). However, there is indeed
evidence of SP-A?Mini-B interactionsmediated bythe micelles.
In zwitterionic micelles, Mini-B demonstrates a strong prefer-
ence to bind SP-A-containing micelles, despite a large excess of
SP-A-free micelles (Figure 4G,H). Furthermore, the DOSY data
Figure 6. Comparison of the average apparent hydrodynamic diameters (dHA) of SP-A in water, pure micelles, individual SP-A? and Mini-B?micelle
complexes, and combined SP-A?Mini-B?micelle complexes as calculated from the 2D DOSY NMR spectra.

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indicate SP-A?Mini-B interactions in all the micelle systems.
For example, with micelles composed of LMPC, LMPG, and
LMPCþLMPG,theapparenthydrodynamicdiameteroftheSP-
A?Mini-B?micelle complex is larger than the Mini-B?micelle
complex but smaller than the SP-A?micelle complex (Table 1
and Figure 6). This may indicate some potentially interesting
effects of Mini-B on SP-A’s oligomeric form in LMPC, LMPG,
and LMPCþLMPG micelles. Also, at least two distinct size
populations of SP-A?Mini-B complexes are found in SDS and
DPC micelles. In DPC, both subpopulations are larger in size
than individual protein?micelle complexes. In SDS, though one
subpopulation is larger, the other one is similar to the size of SP-
A?SDS complex. Therefore, perhaps, the entire populations of
SP-A and Mini-B interact in the presence of DPC micelles but
only subpopulationsof the proteinsinteract inthe presence SDS
micelles. Itispossiblethattheanionicdetergent/lipidmoleculesof
anionicormixedmicellessaturatethecationicsitesoftheremaining
noninteracting Mini-B subpopulation that would otherwise partici-
pate in interactions with the anionic sites of SP-A.
In summary, our work demonstrates the need to revisit the
frequently encountered assumption that SP-A functions as an
octadecamer, since it appears that its lung lipid mimetic micelle-
associated configuration is a smaller oligomeric form. Addition-
ally, we provide evidence for lipid-mediated SP-A?SP-B inter-
actions, which likely contribute to normal lung surfactant func-
tion, and for the ability of SP-B’s structure to be modified by the
compositionoflipidswithwhichitinteracts.Thatthesebehaviors
arefoundtobemodifiedinmicellescomposedofdifferentspecies
butwiththesamechargeunderlinestheimportanceofconsidering
subtleties of protein?lipid interactions, beyond just the electro-
static charge of the lipid headgroups.
’ASSOCIATED CONTENT
b
S
SupportingInformation.
bound micelle fractions; estimation of hydrodynamic diameters
for the SP-A?micelles complexes; comparison of the NMR
signal intensities of SP-A þ Mini-B with Mini-B; prediction of
NMR parameters for different oligomeric forms of SP-A; estima-
tion of masses and oligomeric forms of micelle-bound SP-A;
Tables S1?S5. This material is available free of charge via the
Internet at http://pubs.acs.org.
Estimationoffreeandprotein-
’AUTHOR INFORMATION
Corresponding Author
*Phone (709) 864-4523. Fax (709) 864-2422. E-mail vbooth@
mun.ca.
Funding Sources
Thisresearch wassupported byaCIHROperatingGranttoV.B.
’ACKNOWLEDGMENT
We are particularly grateful to Dr. Alan Waring for providing
the synthetic Mini-B and Mr. Ray Bishop for supplying the cow
lungs.
’ABBREVIATIONS
SP-A, surfactant protein A;SP-B, surfactant protein B;SP-C,
surfactant protein C;CRD, carbohydrate recognition domain;
NMR, nuclear magnetic resonance;SDS, sodium dodecyl sulfate;
DPC, dodecylphosphocholine;LMPC, lysomyristoylphosphati-
dylcholine;LMPG,lysomyristoylphosphatidylglycerol;DPPC,
dipalmitoylphosphatidylcholine;DPPG,dipalmitoylphosphati-
dylglycerol; SDS-PAGE, sodium dodecyl sulfate?polyacryl-
amide gel electrophoresis;MALDI-TOF, matrix-assisted laser
desorption/ionization?time-of-flight;HPLC, high-performance
liquid chromatography;DSS, 2,2-dimethyl-2-silapentane-5-sul-
fonate;DOSY,diffusion-orderedspectroscopy;PFG,pulsedfield
gradient;DC, diffusion coefficient;dHA, apparent hydrodynamic
diameter;TEM, transmission electron microscopy.
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