Kenneth S. Rotondi

A well‐defined amphipathic conformation for the calcium‐free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution

Daptomycin is a 13‐residue cyclic lipopeptide with Ca2+‐dependent bactericidal activity against a variety of high‐risk pathogens. Ring closure in daptomycin is via an ester linkage between the side chain of Thr4 and the C‐terminal carboxyl of the main chain; the N‐terminal residue is capped by a decanoyl aliphatic chain. Extensive NMR data obtained under solution conditions that minimize aggregation have provided constraints for a detailed conformational analysis of daptomycin in aqueous solution, which should facilitate the rational design of improved analogs and enhance understanding of its mode of action. Transannular and shorter‐range nuclear Overhauser effects (NOEs) as well as amide temperature shifts and 3JNHα coupling constants indicate that daptomycin adopts a well‐defined conformation containing a distorted hairpin formed by Gly5–D‐Ala6 type II′ β‐turn. A number of hydrophobic moieties (the lipid N‐cap and the Trp1 and Kyn13 side chains) are clustered at one end of the hairpin, while neutral polar and anionic residues are localized on the other end, leading to amphipathicity in the molecule. These features suggest a mode of action in which the large hydrophobic cluster of the peptide interacts with the acyl chain region of a membrane. This interaction may be facilitated by Ca2+ ions, both by neutralizing the anionic charges and by favoring association with the membrane head groups. Interestingly, our findings differ from two recent articles in which the aqueous conformation of Ca2+‐free daptomycin is reported to lack a well‐defined conformation (D. Jung, A. Rozek, M. Okron, and R. E. W. Hancock, Chemistry & Biology, 2004, Vol. 11, pp. 949–957) or is suggested to populate an alternate conformation (L.‐J. Ball, C. M. Goult, J. A. Donarski, J. Micklefield, and V. Ramesh, Organic & Biomolecular Chemistry, 2004, Vol. 2, pp. 1872–1878).

Native structural propensity in cellular retinoic acid-binding protein I 64–88: the role of locally encoded structure in the folding of a β-barrel protein

A central question in protein folding is the relative importance of locally encoded structure and cooperative interactions among residues distant in sequence. We have been exploring this question in a predominantly beta-sheet protein, since beta-structure formation clearly relies on both local and global sequence information. We present evidence that a 24-residue peptide corresponding to two linked hairpins of cellular retinoic acid-binding protein I (CRABP I) adopts significant native structure in aqueous solution. Prior work from our laboratory showed that the two turns contained in this fragment (turns III and IV) had the highest tendency of any of the eight turns in this anti-parallel beta-barrel to fold into native turns. In addition, the primary sequence of these two turns is well conserved throughout the structural family to which CRABP I belongs, and residues in the turns and their associated hairpins participate in a network of conserved long-range interactions. We propose that the strong local-sequence biases within the chain segment comprising turns III and IV favor longer-range interactions that are crucial to the folding and native-state stability of CRABP I, and may play a similar role in related intracellular lipid-binding proteins (iLBPs).

Local and Long-Range Sequence Contributions to the Folding of a Predominantly β-Sheet Protein

Understanding how a linear sequence of amino acids guides acquisition of the native state of a protein — the protein folding problem — is key to interpreting information present in the human genome and to developing therapeutic approaches to diseases caused by protein misfolding. While considerable progress has been made in elucidating the interplay of local and long-range forces in the folding of α-helical proteins, such understanding has lagged for primarily β-sheet proteins [1]. We have undertaken studies aimed at determining the roles of local and global sequence information in the folding of the predominantly β-sheet protein, cellular retinoic acid binding protein I (CRABP I). CRABP I is a 136 residue β-clamshell protein (Figure 1) whose physiological role is the sequestration and transport of the hydrophobic ligand retinoic acid. CRABP I is a member of the large family of intracellular lipid binding proteins (iLBPs) [2], which contains 52 members with sequence identity greater than 30% with respect to CRABP I. Past work in our laboratory using stopped-flow (SF) mixing to follow the fluorescence of CRABP I has allowed us to define a series of kinetic phases from the urea-denatured conformational ensemble to the native fold [3]. Within the ≈10 ms dead time of the SF instrument, the unfolded ensemble forms a hydro-phobically collapsed state with considerable secondary structure. The presence of significant secondary structure along with hydrophobic collapse suggests that both global and local forces are acting in the earliest folding events.

Conformational Analysis of Human Calcitonin in Aqueous Solution and Comparison with a Highly Potent Lactam-Bridged Analogue

Human calcitonin (hCt) is a peptide hormone of 32 amino acid residues that plays a central role in calcium metabolism [1,2]. The receptor-bound conformation of hCt has not yet been determined. In an effort to understand the bioactive conformation of hCt we have recently designed a lactam-bridged hCt analogue with the structure cyclo17,21-[Asp17,Orn21]hCt (1) and found it to be 400-times more potent than hCt and 4-times more potent than salmon Ct (sCt) in the in vivo hypocalcemic assay in mice, making this the most potent known hCt analogue [3]. By contrast, a linear control peptide [Asp17, Orn21]hCt (2) showed similar in vivo potency to hCt [3]. These results suggested that the conformation of 1 may correspond to a bioactive conformation of hCt [3]. Here, we present studies on the solution conformation of hCt and 1 based on NMR in combination with molecular modeling and CD spectroscopy. We also present data on the hCt receptor-binding affinity of 1 as compared to 2, hCt and sCt.

Using peptides to study the folding of a predominantly β-sheet protein

While the primary sequence of a protein is sufficient to guide it to its folded state under native conditions from an ensemble of unfolded conformations in the presence of denaturant, we do not yet know the relative importance of local sequence-directed conformational preferences and global information such as the pattern of hydrophobic and hydrophilic residues. We have described the folding pathway of cellular retinoic acid binding protein I (CRABPI), a predominantly β -sheet protein with an internal cavity that binds retinoic acid, based on results from a variety of methods (stopped flow fluorescence and circular dichroism, quenched flow hydrogen exchange) [1,2]: An early kinetic phase (< 10 msec) appears to correspond to hydrophobic collapse and is accompanied by development of substantial native-like CD signal. The topology of the protein including presence of the central ligand binding cavity develops in a 100 msec kinetic phase [2]. Intriguingly, stable hydrogen bonding in the β sheets is present only after a longer kinetic phase (1 sec), which is also accompanied by the development of native tertiary interactions. A minor population of the molecules folds more slowly (15 to 20 sec) due to slow cis-trans isomerization around X -Pro bonds. We are now examining the importance of local sequence in this folding pathway in two ways: 1) by studies of peptide fragments; and 2) by studies of mutated versions of CRABPI. Thus far, we have found that the small helix–turn–helix of CRABPI has a strong locally driven folding tendency including a sequence motif called a Schellman motif [3]. In the present study, we have focused on the roles that local sequences in turns may play in the folding of CRABPI. The topology of a β -sheet protein requires that non-local segments of chain make specific contacts, yet local sequence must play a role early in folding to favor these interactions. The turns connecting strands are likely to facilitate the formation of the adjacent strand-strand contacts. In CRABPI, like many of its family members, tight turns connect most of the β strands. We have examined characteristics of the tight turns in CRABPI that may point out key local sequences for folding. If a turn is involved in guiding the chain towards the native topology, then one would expect it to be stabilized largely by interactions within its own sequence, even in the context of the native protein