Effect of chain topology on the self-organization and dynamics of block copolypeptides: from diblock copolymers to stars

The effect of chain topology on (i) the peptide secondary structure, (ii) the nanophase self-assembly, and (iii) the local segmental and global peptide relaxations has been studied in a series of model diblock and 3-arm star copolypeptides of poly(epsilon-carbobenzyloxy-L-lysine) (PZLL) and poly(gamma-benzyl-L-glutamate) (PBLG) with PZLL forming the core. Diblock copolypeptides are nanophase separated with PBLG and PZLL domains comprising alpha-helices packed in a hexagonal lattice. Star copolypeptides are only weakly phase separated, comprising PBLG and PZLL alpha-helices in a pseudohexagonal lattice. Phase mixing has profound consequences on the local and global dynamics. The relaxation of the peptide secondary structure speeds up, and the helix persistence length is further reduced in the stars, signifying an increased concentration of helical defects.     

Molecular blending by polymerization of intercalated solvent. Poly(gamma-benzyl-L-glutamate)/benzyl methacrylate as a model system

The aim of the present research is to obtain blending between a polymer and a (polymerized) solvent on the molecular level. Because of its rigid rod structure, poly(gamma-benzyl-L-glutamate) (PBLG) is chosen as the polymer. Benzyl methacrylate (BzMA) has been chosen as the solvent for two reasons. First, the structure of the solvent is very similar to the structure of the side chain of PBLG, favoring interactions between the two materials. Second, the solvent can be polymerized, because of the presence of a C=C bond. In cast films of PBLG and BzMA separate zones of the polymer and solvent are present. Wide-angle X-ray diffraction and Raman results show that upon heating the cast films homogenization occurs and solvent molecules intercalate between the helices of PBLG. At 150 degrees C a hexagonal packing is obtained. The dimensions of the obtained packing depend on the solvent concentration, which confirms that solvent molecules are indeed present within the crystalline lattice. DSC experiments imply that the observed changes upon heating correspond to thermodynamic processes. On cooling the homogeneous samples, disordering of the hexagonal packing occurs. Polymerization of the homogeneous samples results in a disordering of the hexagonal packing and in a contraction of the unit cell. The latter once more confirms that solvent molecules are indeed present within the crystalline lattice. The applied principle of polymerization of a solvent in a molecular homogeneous system can be favorable for many applications, for which morphology control at the molecular level is required.     

Model peptide studies of sequence regions in the elastomeric biomineralization protein, Lustrin A. I. The C-domain consensus-PG-, -NVNCT-motif

The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin‐containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force‐extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force‐induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12‐residue consensus sequence found near the N‐termini of the first eight cysteine‐rich domains (C‐domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12‐mer sequence adopts a loop conformation, consisting of a “bend” or “turn” involving residues G3–K4 and N7–C8–T9, with extended conformations arising at F1–G3; K4–V6; T9–S10–G11 in the sequence. Minor pH‐dependent conformational effects were noted for this peptide; however, there is no evidence for a salt‐bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved —PG—, —NVNCT— sequence of C1–C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior. © 2002 Wiley Periodicals, Inc. Biopolymers 63: 358–369, 2002     

Thermodynamic confinement and alpha-helix persistence length in poly(gamma-benzyl-L-glutamate)-b-poly(dimethyl siloxane)-b-poly(gamma-benzyl-L-glutamate) triblock copolymers

The structure and the associated dynamics of a series of poly(gamma-benzyl-L-glutamate)-b-poly(dimethyl siloxane)-b-poly(gamma-benzyl-L-glutamate) (PBLG-b-PDMS-b-PBLG) triblock copolymers were investigated using small- and wide-angle X-ray scattering, NMR, transmission electron microscopy, and dielectric spectroscopy, respectively. The structural analysis revealed phase separation in the case of the longer blocks with defected alpha-helical segments embedded within the block copolymer nanodomains. The alpha-helical persistence length was found to depend on the degree of segregation; thermodynamic confinement and chain stretching results in the partial annihilation of helical defects.     

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T相似文献   

Spatial variation in branch size promotes metapopulation persistence in dendritic river networks

1. Despite years of attention, the dynamics of species constrained to disperse within riverine networks are not well captured by existing metapopulation models, which often ignore local dynamics within branches.
2. We develop a modelling framework, based on traditional metapopulation theory, for patch occupancy dynamics subject to local colonisation–extinction dynamics within branches and regional dispersal between branches in size-structured, bifurcating riverine networks. Using this framework, we investigate whether and how spatial variation in branch size affects species persistence for dendritic systems with directional dispersal, including one-way (up- or downstream only) and two-way (both up- and downstream) dispersal.
3. Variation in branch size generally promotes species persistence more obviously at higher relative extinction rate, suggesting that previous studies ignoring differences in branch size in real riverine systems might overestimate species extinction risk.
4. Two-way dispersal is not always superior to one-way dispersal as a strategy for metapopulation persistence especially at high relative extinction rate. The type of dispersal that maximises species persistence is determined by the hierarchical level of the largest, and hence most influential, branch within the network. When considering the interactive effects of up- and downstream dispersal, we find that moderate upstream-biased dispersal maximises metapopulation viability, mediated by spatial branch arrangement.
5. Overall, these results suggest that both branch-size variation and species traits interact to determine species persistence, theoretically demonstrating the ecological significance of their interplay.

     

On the interactions of sensitive and resistant Mycobacterium tuberculosis to antibiotics

In this work we propose a system of non linear ordinary differential equations for the dynamics of Mycobacterium tuberculosis (Mtb) within the host, in order to study the role of macrophages, T cells and antibiotics in the control of sensitive and resistant Mtb. Conditions for the persistence of sensitive and resistant bacteria are given in terms of the secondary infections produced by bacteria and macrophages, the immune response, and the antibiotic treatment. Model analysis predicts backward bifurcations for certain values of the parameters. In this case, the dynamics is characterized by the coexistence of two infection states with low and high bacteria load, respectively.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.     

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid β peptide involved in Alzheimer's disease. We focused our study on the Glu²² residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and β-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu²² in wild-type) or hydrophobic (Val²² in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and α-helix structures (with low β-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Aβ_12-28 variants does not correlate with the amount of β-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.     

Order within disorder: Aggrecan chondroitin sulphate‐attachment region provides new structural insights into protein sequences classified as disordered

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)‐attachment region of human aggrecan (CS‐peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS‐peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS‐peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small‐angle X‐ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2‐G3 construct. The semiordered structure identified in CS‐peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of the sequence and size of non-polar residues on self-assembly of amphiphilic peptides

Peptides with alternating hydrophobic and polar amino acids have been shown to form stable beta-sheet secondary structures and self-assemble into hydrogel-like matrices in the presence of physiological salt concentrations. We hypothesized that the sequence and steric size differences of non-polar residues can affect the balance of peptide intermolecular forces in solution that drive self-assembly. To test this hypothesis, we designed a library of artificial amphiphilic peptides based on the sequence (FEFEFKFK)2 by substituting combinations of the non-polar residues glycine, alanine, valine, leucine and isoleucine for phenylalanine. Peptide structure and self-assembly were characterized using scanning electron microscopy, the Thioflavin T assay, transmission electron microscopy, X-ray fiber diffraction and circular dichroism spectroscopy. The sequence and steric size of non-polar residues are shown to cause variations in peptide secondary structures and create significant differences in the matrix morphology of self-assembled peptides.     

Emergence of large-scale cell morphology and movement from local actin filament growth dynamics

Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged “canoe” shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features—cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence—that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes—which had VASP highly enriched at their leading edges and migrated fast with straight trajectories—to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions.     

Solution properties of synthetic polypeptides. XVII. Stability of the alpha-helical conformation of poly (gamma-benzyl L-glutamate) in helicogenic solvents

The Zimm–Bragg parameters s and σ were determined for poly(γ-benzyl L -glutamate) (PBLG) in m-cresol and in dimethylformamide (DMF) from ORD data as a function of molecular weight. It was found that, within the temperature range between 10 and 55°C and on the average, s = 1.6₁ ± 0.1 and √σ = 0.04 ± 0.01 in m-cresol and s = 1.6₅ ± 0.05 and √σ = 0.045 ± 0.015 in DMF. The values of s in m-cresol decreased with increasing temperature, while the values of σ in the same solvent increased. This result for s suggests that PBLG in m-cresol will undergo a thermal helix–coil transition of normal type. The parameters in DMF showed no appreciable trend to vary with temperature. Aside from the difference between the two solvents, our results are consistent with existing data for various conformation-dependent properties such as light-scattering radius, intrinsic viscosity, and dipole moment, each indicating that the polypeptide chain has some flexibility in helicogenic solvents.     

Crowding and confinement effects on protein diffusion in vivo

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, , and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, , in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/ (standard deviation, sigma(D), relative to the mean), is 0.39. Both and sigmaD/ remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in by a factor of 380 over the range of Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/. Individual values of D vary by a factor of 9 x 10(4) but correlate well with f(VPS), the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta values related to variation in f(VPS). Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.     

Structural, stability, dynamic and binding properties of the ALS-causing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.     

Toward intrinsically fluorescent proteomimetics: fluorescent probe response to alpha helix structure of poly-gamma-benzyl-L-glutamate

A fluorescent probe (1), developed for recognition of alpha helical secondary structure, shows a large fluorescence change upon titration with the synthetic protein PBLG. Compared to fluorophores of similar size and shape, 1 displayed the smallest dissociation constant (K(D)=80microM) when titrated with PBLG. These preliminary studies are directed toward developing small molecule proteomimetics that have intrinsic fluorescence and are specific for helical-protein binding-sites.     

Subcutaneous Construction of Engineered Adipose Tissue with Fat Lobule-Like Structure Using Injectable Poly-Benzyl-L-Glutamate Microspheres Loaded with Adipose-Derived Stem Cells

Porous microcarriers were fabricated from synthesized poly(γ-benzyl-L-glutamate) (PBLG) polymer to engineer adipose tissue with lobule-like structure via the injectable approach. The adipogenic differentiation of human adipose-derived stem cells (hASCs) seeded on porous PBLG microcarriers was determined by adipogenic gene expression and glycerol-3-phosphate dehydrogenase enzyme activity. In vitro adipogenic cultivation was performed for 7 days, and induced hASC/PBLG complex (Adi-ASC/PBLG group) was subcutaneously injected into nude mice. Injections of PBLG microcarriers alone (PBLG group) and non-induced hASC/PBLG complex (ASC/PBLG group) served as controls. Newly formed tissues were harvested after 4 and 8 weeks. Generation of subcutaneous adipose tissue with typical lobule-like structure separated by fibrous septa was observed upon injection of adipogenic-induced hASC/microsphere complex. Adipogenesis significantly increased in the Adi-ASC/PBLG group compared with the control groups. The angiogenesis in the engineered adipose tissue was comparable to that in normal tissue as determined by capillary density and luminal diameter. Cell tracking assay demonstrated that labeled hASCs remained detectable in the neo-generated tissues 8 weeks post-injection using green fluorescence protein-labeled hASCs. These results indicate that adipose tissue with typical lobule-like structure could be engineered using injectable porous PBLG microspheres loaded with adipogenic-induced hASCs.     

Three-dimensional structure and dynamics of wine tannin-saliva protein complexes. A multitechnique approach

The interactions between the B3 (catechin-4alpha,8-catechin) red wine tannin and the human salivary protein fragment IB7(14) (SPPGKPQGPPPQGG) were monitored by (1)H magic angle spinning NMR, circular dichroism, electrospray ionization mass spectrometry, and molecular modeling. It is found that the secondary structure of IB7(14) is made of a type II helix (collagen helix) and random coil. The central glycine 8 appears to act as a flexible rotula separating two helix II regions. Three tannin molecules tightly complex the peptide, without modifying its secondary structure, but seem to reduce its conformational dynamics. The binding dissociation constant is in the millimolar range. B3 tannins with a "tweezers" conformation bind to the hydrophilic side of the saliva peptide, suggesting that the principal driving forces toward association are governed by hydrogen bonding between the carbonyl functions of proline residues and both the phenol and catechol OH groups. These findings are further discussed in the frame of an astringency phenomenon.     

Dynamic Functional Modulation of CD4+ T Cell Recall Responses Is Dependent on the Inflammatory Environment of the Secondary Stimulus

The parameters that modulate the functional capacity of secondary Th1 effector cells are poorly understood. In this study, we employ a serial adoptive transfer model system to show that the functional differentiation and secondary memory potential of secondary CD4⁺ effector T cells are dependent on the inflammatory environment of the secondary challenge. Adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus (LCMV) Glycoprotein-specific SMARTA memory cells into LCMV-immune hosts, followed by secondary challenge with Listeria monocytogenes recombinantly expressing a portion of the LCMV Glycoprotein (Lm-gp61), resulted in the rapid emergence of SMARTA secondary effector cells with heightened functional avidity (as measured by their ability to make IFNγ in response to ex vivo restimulation with decreasing concentrations of peptide), limited contraction after pathogen clearance and stable maintenance secondary memory T cell populations. In contrast, transfer of SMARTA memory cells into naïve hosts prior to secondary Lm-gp61 challenge, which resulted in a more extended infectious period, resulted in poor functional avidity, increased death during the contraction phase and poor maintenance of secondary memory T cell populations. The modulation of functional avidity during the secondary Th1 response was independent of differences in antigen load or persistence. Instead, the inflammatory environment strongly influenced the function of the secondary Th1 response, as inhibition of IL-12 or IFN-I activity respectively reduced or increased the functional avidity of secondary SMARTA effector cells following rechallenge in a naïve secondary hosts. Our findings demonstrate that secondary effector T cells exhibit inflammation-dependent differences in functional avidity and memory potential, and have direct bearing on the design of strategies aimed at boosting memory T cell responses.