Partitioning, dynamics, and orientation of lung surfactant peptide KL(4) in phospholipid bilayers.

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL(4) is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL(4) 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 KL(4) in phospholipid bilayers, we introduced CD(3)-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via (2)H 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 KL(4) in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL(4) 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 KL(4) relative to antimicrobial amphipathic alpha-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 KL(4). The unusual secondary structure of KL(4) and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

Kinetics of folding and binding of an intrinsically disordered protein: the inhibitor of yeast aspartic proteinase YPrA.

The 68 residue peptide IA 3 is an intrinsically unstructured protein that serves as an endogenous inhibitor of the yeast aspartic proteinase A (YPrA). Although unstructured in free solution, IA 3 forms an N-terminal alpha helix as it binds to YPrA, leading to subnanomolar inhibition of the protease. Equilibrium structural and inhibition studies provide little insight into the mechanism and kinetics of the coupled folding and binding interaction. We have used laser temperature jump spectroscopy to study the kinetics of folding of free IA 3 and of the interaction between IA 3 and YPrA. Inducing folding with trifluoroethanol cosolvent allows us to determine the folding rate (kf approximately 0.3 (micros)(-1)) and the unfolding rate (ku approximately 3 (micros)(-1)) for free IA 3 in water at 25 degrees C. A substantially faster relaxation process is observed in the presence of the proteinase; this process appears to be the kinetic signature of an intermediate binding step in the coupled folding and binding interaction of IA 3 and YPrA.

The helical structure of surfactant peptide KL4 when bound to POPC: POPG lipid vesicles.

KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B, which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic alpha-helix; qualitative measurements using Raman, CD, and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing (13)C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (phi, psi) angles that differ substantially from common values for alpha-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC:POPG lipid vesicles are (-105, -30); this deviation from ideal alpha-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.

Palaeotemperature trend for Precambrian life inferred from resurrected proteins.

Biosignatures and structures in the geological record indicate that microbial life has inhabited Earth for the past 3.5 billion years or so. Research in the physical sciences has been able to generate statements about the ancient environment that hosted this life. These include the chemical compositions and temperatures of the early ocean and atmosphere. Only recently have the natural sciences been able to provide experimental results describing the environments of ancient life. Our previous work with resurrected proteins indicated that ancient life lived in a hot environment. Here we expand the timescale of resurrected proteins to provide a palaeotemperature trend of the environments that hosted life from 3.5 to 0.5 billion years ago. The thermostability of more than 25 phylogenetically dispersed ancestral elongation factors suggest that the environment supporting ancient life cooled progressively by 30 degrees C during that period. Here we show that our results are robust to potential statistical bias associated with the posterior distribution of inferred character states, phylogenetic ambiguity, and uncertainties in the amino-acid equilibrium frequencies used by evolutionary models. Our results are further supported by a nearly identical cooling trend for the ancient ocean as inferred from the deposition of oxygen isotopes. The convergence of results from natural and physical sciences suggest that ancient life has continually adapted to changes in environmental temperatures throughout its evolutionary history.

Characterizing the residue level folding of the intrinsically unstructured IA3.

Residue level analysis of the folding of simple proteins may hold the key to understanding folding pathways and aid in structure prediction. IA(3), the endogenous inhibitor of yeast aspartic proteinase A (YPrA), is an unstructured protein in solution. Comparison of the 2D (15)N-HSQC spectra of IA(3) in water and in 23% 2,2,2-trifluoroethanol (TFE) shows that the individual residue cross peaks of IA(3) become more dispersed in the presence of TFE, indicating that the protein undergoes an unstructured to structured transition in the presence of TFE. This transition can be monitored by the movements of the cross peaks. Following the individual cross peaks, however, is complicated and does not establish whether a single transition occurs globally in the sequence. In this equilibrium study, we apply singular value decomposition (SVD) to elucidate both the main features of the TFE-driven transition and the residue-level deviations from the average behavior. This analysis has yielded a two-state folding description as well as specifics of NMR frequency shifts of individual residues, indicating that the N-terminus of IA(3) has a higher helical propensity than the C-terminus. Additionally, we discuss possible mechanisms for observed deviations from a two-state folding transition. When combined with a traditional biochemical understanding of interactions between individual residues, this approach leads to a better understanding of protein folding.