HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence.

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with 125I-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitism of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.

Variability of a bacterial surface protein and disease expression in a possible mouse model of systemic Lyme borreliosis.

During persistent infection of scid mice with Borrelia turicatae, an agent of relapsing fever and neuroborreliosis, there was variation in the surface proteins the bacteria expressed and in disease manifestations over time. Two serotypes, A and B, were isolated from the mice, cloned by limiting dilution, and further characterized. The only discernible difference between the two variants was in the size of the major surface protein they expressed: serotype A had a variable major protein (Vmp) of 23,000, and serotype B had a Vmp of 20,000. When other scid mice were inoculated with clonal populations of A and B, the infections were similar with respect to onset and degree of spirochetemia, involvement of the eye and heart, and occurrence of a peripheral vestibular disorder. However, there were differences between the serotypes in other respects: (a) serotype B but not A caused reddened and significantly enlarged joints, markedly impaired performance on a walking bar, and severe arthritis by histologic examination; (b) serotype A but not B invaded the central nervous system during early infection; and (c) serotype A penetrated monolayers of human umbilical vein endothelial cells more readily than did serotype B. The combination of arthritis, myocarditis, and neurologic disease resembled human Lyme borreliosis. The findings indicate that differences in disease expression are determined by variable surface proteins of the bacterium and that scid mouse infections with B. turicatae provide a model for the study of the pathogenesis of Lyme borreliosis and other persistent spirochetal diseases.

Fibronectin mediates Treponema pallidum cytadherence through recognition of fibronectin cell-binding domain.

The specificity of the interaction between Treponema pallidum and fibronectin was demonstrated. Treatment of host cells with only antifibronectin sera and not anticollagen or antilaminin sera, inhibited treponemal cytadsorption. Incubation of fibronectin-coated coverslips with monoclonal antibody to the cell-binding domain of fibronectin reduced treponemal attachment to the same extent as antifibronectin serum. Both iodinated fibronectin and iodinated cell-binding domain bound to T. pallidum in a saturable manner. Specificity of the T. pallidum association with the cell-binding domain was the most effective inhibitor of the binding of either radioiodinated cell-binding domain or fibronectin to T. pallidum. Scatchard analysis gave Kd on the order of 10(-7) M for both cell-binding domain and fibronectin binding to T. pallidum, consistent with the high affinity interaction of these organisms with host cell surfaces. Finally, the same level of attachment of treponemes was achieved on coverslips coated with cell-binding domain as that observed for organisms incubated with fibronectin, indicating that the cell-binding domain polypeptide is functionally identical to fibronectin in mediating T. pallidum adherence.

Insect population control using a dominant, repressible, lethal genetic system.

A major modification to the sterile insect technique is described, in which transgenic insects homozygous for a dominant, repressible, female-specific lethal gene system are used. We demonstrate two methods that give the required genetic characteristics in an otherwise wild-type genetic background. The first system uses a sex-specific promoter or enhancer to drive the expression of a repressible transcription factor, which in turn controls the expression of a toxic gene product. The second system uses non-sex-specific expression of the repressible transcription factor to regulate a selectively lethal gene product. Both methods work efficiently in Drosophila melanogaster, and we expect these principles to be widely applicable to more economically important organisms.

A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization.

A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.

Effects of Ser16 phosphorylation on the allosteric transitions of phospholamban/Ca(2+)-ATPase complex.

Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions.

An electron paramagnetic resonance study of skeletal muscle membrane fluidity in malignant hyperthermia.

Skeletal muscle sarcolemma (SL), transverse tubule (TT) and heavy sarcoplasmic reticulum (HSR) membranes were isolated from malignant hyperthermia susceptible (MHS) and normal pigs, and the rotational dynamics of lipid hydrocarbon chain motion was examined by electron paramagnetic resonance (EPR) spectroscopy. The stearic acid spin probe 16-SASL was incorporated into MHS and normal membranes and both the order parameter (S) and effective correlation time (tau r) of probe motion were calculated from spectra recorded over the temperature range of 2 to 40 degrees C. At any given temperature, TT membranes exhibited significantly greater values for both the S and tau r of probe motion than did SL, which exhibited significantly greater values than did HSR membranes. The order of decreasing S and tau r values for 16-SASL mobility correlated with the decreasing cholesterol content of these membranes (TT greater than SL greater than HSR), however there was no difference in the S or tau r values for a given membrane fraction isolated from both MHS and normal muscle. Arrhenius plots of 16-SASL mobility in SL, TT and HSR were linear from 2 to 40 degrees C, indicating no abrupt thermotropic change in the lipid hydrocarbon phase of any of the membrane types studied. Apparent activation energies (Ea), calculated from the Arrhenius plots, were similar for MHS and normal membranes derived from a given cellular location. However, the Ea of probe motion for TT membranes (2.3 +/- 0.1 and 2.4 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) was significantly less than for SL (3.4 +/- 0.4 and 2.9 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) which, in turn, was significantly less than the Ea for HSR (3.7 +/- 0.1 and 3.7 +/- 0.1 kcal/mol/degree for MHS and normal, respectively). Since 16-SASL motion was similar in MHS and normal membranes, we conclude that there is no evidence for a generalized membrane defect affecting lipid mobility in these MHS muscle membranes.

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length.

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.

Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay.

In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.

Ionic effects on the rotational dynamics of cross-bridges in myosin filaments, measured by triplet absorption anisotropy.

We have measured the rotational motion of myosin heads in synthetic thick filaments at 4 degrees C in the time range from 10(-7) to 10(-4) seconds, by measuring transient absorption anisotropy of an eosin probe attached to a reactive sulfhydryl on the myosin head. Under conditions that result in monomeric myosin (500 mM ionic strength), the anisotropy decay is independent of pH in the range from 7.0 to 8.2 and [Mg2+] in the range from 0.1 to 10 mM; the anisotropy decays bi-exponentially with correlation times of 0.4 and 2 microseconds to a constant value of 0.016. Under more physiological conditions (115 mM ionic strength), resulting in filament formation, the anisotropy decay is sensitive to both pH and [Mg2+]. The anisotropy at pH 8.2 and 0.1 mM-Mg2+ decays with correlation times of 0.5 and 3.8 microseconds to a constant limiting anisotropy of 0.038. When the [Mg2+] is increased to 10 mM, the correlation times are 0.6 and 5.7 microseconds and the limiting anisotropy value is 0.055. Identical changes in the anisotropy decay are caused by an increase in [H+] to pH 7.0, in the presence of 0.1 mM-Mg2+. Increasing the total ionic strength to 187 mM decreases the amplitude of the cation effects. These results provide direct evidence that the rotational dynamics of myosin heads in thick filaments are influenced by physiological concentrations of cations. The results are qualitatively consistent with the proposal that these and other ionic conditions regulate transitions between "spread" and "compact" cross-bridge conformations, but the quantitative results indicate that cross-bridges undergo large-amplitude microsecond rotations even under conditions where the compact state should predominate.

Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer.

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for Förster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.

Cooperativity in F-actin: binding of gelsolin at the barbed end affects structure and dynamics of the whole filament.

We have studied the effect of gelsolin, a Ca-dependent actin-binding protein, on the microsecond rotational dynamics of actin filaments, using time-resolved phosphorescence (TPA) and absorption anisotropy (TAA) of erythrosin iodoacetamide attached to Cys374 on actin. Polymerization of actin in the presence of gelsolin resulted in substantial increases in the rate and amplitude of anisotropy decay, indicating increased rotational motion. Analysis indicates that the effect of gelsolin cannot be explained by increased rates of overall (rigid-body) rotations of shortened filaments, but reflects changes in intra-filament structure and dynamics. We conclude that gelsolin induces (1) a 10 degrees change in the orientation of the absorption dipole of the probe relative to the actin filament, indicating a conformational change in actin, and (2) a threefold decrease in torsional rigidity of the filament. This result, which is consistent with complementary electron microscopic observations on the same preparations, directly demonstrates long-range cooperativity in F-actin, where a conformational change induced by the binding of a single gelsolin molecule to the barbed end is propagated along inter-monomer bonds throughout the actin filament.

Microsecond rotational dynamics of actin: spectroscopic detection and theoretical simulation.

We have investigated the microsecond rotational dynamics of F-actin with transient phosphorescence anisotropy (TPA) spectroscopy, and analyzed the data to determine the relative contributions from rigid-body rotations and from intrafilament bending and twisting, using a theoretical model developed for DNA dynamics by Schurr and co-workers. The fits of the data to the model were constrained by independently determining the orientation of the dye's absorption dipole (by transient absorption anisotropy, TAA) and the actin filament length distribution (by electron microscopy). We conclude that (1) the Schurr theory enables calculation of the torsional flexibility of actin independent of any contribution from rigid body rotations of the whole filament, (2) the TPA decays cannot be explained by rigid-body or bending rotations, but reflect primary twisting motions within actin filaments, and (3) the dynamic properties of actin filaments are best ascribed to a continuous elasticity. This analysis establishes a firm methodological foundation for future studies of the effects or perturbations of the dynamics of actin on its functional properties.

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence.

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanochemical coupling in spin-labeled, active, isometric muscle.

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force? Pate and Cooke (1989. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.

Direct spectroscopic detection of molecular dynamics and interactions of the calcium pump and phospholamban.

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.

Oculoplethysmography/carotid phonoangiography: its value as a screening test in patients with suspected carotid artery stenosis.

A review of 291 carotid arteries that were subjected to both oculoplethysmography/carotid phonoangiography (OPG/CPA) and arteriography showed that although 84% of the vessels that were narrowed less than 40% were properly categorized by the noninvasive test, there was poor grading ability for lesions 40% or greater, and an unacceptably high (40%) false-negative rate for marked carotid stenoses. This has been confirmed by others. Analysis of a 15-item questionnaire that was returned by 12 clinicians showed an unwarranted reliance on the OPG/CPA as a screening test in those patients without transient ischemic attacks. A large majority of responding physicians favored endarterectomy in asymptomatic patients with lesions greater than 70%, and the use of OPG/CPA prevented a substantial number of affected individuals from undergoing angiography and subsequent corrective surgery.

Growth potential of the transplanted lung in the infant primate.

Success in neonatal lung transplantation depends on the growth of the transplanted lung. To study the effects of transplantation and denervation on primate lung growth without rejection or immunosuppression, an autotransplant model was chosen. Eight-week-old baboons underwent left lung autotransplantation (n = 5) or sham operation (n = 1). At age 13 weeks and 9 months, single lung volumes were calculated by nitrogen washout and computed tomography. Results were compared with those of 4 unoperated weight-matched controls (2 per age group). Over the growth period, mean total lung capacity in operated baboons increased 82% (137 to 249 mL) by nitrogen washout and 70% (182 to 309 mL) by computed tomography compared with 85% (128 to 237 mL) and 74% (141 to 245 mL) for the sham-operated baboon, respectively. Transplanted left lung volume increased 91% (53 to 101 mL) by nitrogen washout and 75% (68 to 119 mL) by computed tomography compared with 85% (54 to 100 mL) and 80% (56 to 101 mL) for the sham-operated baboon, respectively. In the absence of rejection and immunosuppression, normal volume growth occurs in the transplanted infant primate lung.