High-density lipoprotein maintains skeletal muscle function by modulating cellular respiration in mice.

BACKGROUND: Abnormal glucose metabolism is a central feature of disorders with increased rates of cardiovascular disease. Low levels of high-density lipoprotein (HDL) are a key predictor for cardiovascular disease. We used genetic mouse models with increased HDL levels (apolipoprotein A-I transgenic [apoA-I tg]) and reduced HDL levels (apoA-I-deficient [apoA-I ko]) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle. METHODS AND RESULTS: ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test compared with wild-type mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved glucose tolerance test, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of endurance capacity compared with wild-type mice. Circulating levels of fibroblast growth factor 21, a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high-fat diet-induced impairment of glucose homeostasis. CONCLUSIONS: In view of impaired mitochondrial function and decreased HDL levels in type 2 diabetes mellitus, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of type 2 diabetes mellitus beyond cardiovascular disease.

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins.

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.

High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli.

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.

Hepatic deficiency of low density lipoprotein receptor-related protein-1 reduces high density lipoprotein secretion and plasma levels in mice.

The low density lipoprotein receptor-related protein-1 (LRP1) is known to serve as a chylomicron remnant receptor in the liver responsible for the binding and plasma clearance of apolipoprotein E-containing lipoproteins. Previous in vitro studies have provided evidence to suggest that LRP1 expression may also influence high density lipoprotein (HDL) metabolism. The current study showed that liver-specific LRP1 knock-out (hLrp1(-/-)) mice displayed lower fasting plasma HDL cholesterol levels when compared with hLrp1(+/+) mice. Lecithin:cholesterol acyl transferase and hepatic lipase activities in plasma of hLrp1(-/-) mice were comparable with those observed in hLrp1(+/+) mice, indicating that hepatic LRP1 inactivation does not influence plasma HDL remodeling. Plasma clearance of HDL particles and HDL-associated cholesteryl esters was also similar between hLrp1(+/+) and hLrp1(-/-) mice. In contrast, HDL secretion from primary hepatocytes isolated from hLrp1(-/-) mice was significantly reduced when compared with that observed with hLrp1(+/+) hepatocytes. Biotinylation of cell surface proteins revealed decreased surface localization of the ATP-binding cassette, subfamily A, member 1 (ABCA1) protein, but total cellular ABCA1 level was not changed in hLrp1(-/-) hepatocytes. Finally, hLrp1(-/-) hepatocytes displayed reduced binding capacity for extracellular cathepsin D, resulting in lower intracellular cathepsin D content and impairment of prosaposin activation, a process that is required for membrane translocation of ABCA1 to facilitate cholesterol efflux and HDL secretion. Taken together, these results documented that hepatic LRP1 participates in cellular activation of lysosomal enzymes and through this mechanism, indirectly modulates the production and plasma levels of HDL.

Structure of apolipoprotein A-I in spherical high density lipoproteins of different sizes.

Spherical high density lipoproteins (HDL) predominate in human plasma. However, little information exists on the structure of the most common HDL protein, apolipoprotein (apo) A-I, in spheres vs. better studied discoidal forms. We produced spherical HDL by incubating reconstituted discoidal HDL with physiological plasma-remodeling enzymes and compared apoA-I structure in discs and spheres of comparable diameter (79-80 and 93-96 A). Using cross-linking chemistry and mass spectrometry, we determined that the general structural organization of apoA-I was overall similar between discs and spheres, regardless of diameter. This was the case despite the fact that the 93 A spheres contained three molecules of apoA-I per particle compared with only two in the discs. Thus, apoA-I adopts a consistent general structural framework in HDL particles-irrespective of shape, size and the number of apoA-Is present. Furthermore, a similar cross-linking pattern was demonstrated in HDL particles isolated from human serum. We propose the first experiment-based molecular model of apoA-I in spherical HDL particles. This model provides a new foundation for understanding how apoA-I structure modulates HDL function and metabolism.

Speciated human high-density lipoprotein protein proximity profiles.

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (∼58%), phospholipids (∼21%), total cholesterol (∼16%), triglycerides (∼5%), and free cholesterol (∼4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.

Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.

OBJECTIVE: Recent proteomic studies have identified multiple proteins that coisolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s). METHODS AND RESULTS: We investigated the distribution of proteins across 5 physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more in-depth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1, and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation. CONCLUSIONS: These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.

Xanthophylls, phytosterols and pre-ß1-HDL are differentially affected by fenofibrate and niacin HDL-raising in a cross-over study.

Fenofibrate and extended-release (ER) niacin similarly raise high-density lipoprotein cholesterol (HDL-C) concentration but their effects on levels of potent plasma antioxidant xanthophylls (lutein and zeaxanthin) and phytosterols obtained from dietary sources, and any relationship with plasma lipoproteins and pre-ß1-HDL levels, have not been investigated. We studied these parameters in 66 dyslipidemic patients treated for 6 week with fenofibrate (160 mg/day) or ER-niacin (0.5 g/day for 3 week, then 1 g/day) in a cross-over study. Both treatments increased HDL-C (16 %) and apolipoprotein (apo) A-I (7 %) but only fenofibrate increased apoA-II (28 %). Lutein and zeaxanthin levels were unaffected by fenofibrate but inversely correlated with percentage change in apoB and low-density lipoprotein cholesterol and positively correlated with end of treatment apoA-II. ApoA-II in isolated HDL in vitro bound more lutein than apoA-I. Xanthophylls were increased by ER-niacin (each ~30 %) without any correlation to lipoprotein or apo levels. Only fenofibrate markedly decreased plasma markers of cholesterol absorption; pre-ß1-HDL was significantly decreased by fenofibrate (-19 %, p < 0.0001), with little change (3.4 %) for ER-niacin. Although fenofibrate and ER-niacin similarly increased plasma HDL-C and apoA-I, effects on plasma xanthophylls, phytosterols and pre-ß1-HDL differed markedly, suggesting differences in intestinal lipidation of HDL. In addition, the in vitro investigations suggest an important role of plasma apoA-II in xanthophyll metabolism.

Apolipoprotein A-I structural organization in high-density lipoproteins isolated from human plasma.

High-density lipoproteins (HDLs) mediate cholesterol transport and protection from cardiovascular disease. Although synthetic HDLs have been studied for 30 years, the structures of human plasma-derived HDL and its major protein apolipoprotein apoA-I are unknown. We separated normal human HDL into five density subfractions and then further isolated those containing predominantly apoA-I (LpA-I). Using cross-linking chemistry and mass spectrometry, we found that apoA-I adopts a structural framework in these particles that closely mirrors that in synthetic HDL. We adapted established structures for synthetic HDL to generate the first detailed models of authentic human plasma HDL in which apoA-I adopts a symmetrical cage-like structure. The models suggest that HDL particle size is modulated by means of a twisting motion of the resident apoA-I molecules. This understanding offers insights into how apoA-I structure modulates HDL function and its interactions with other apolipoproteins.

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles.

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.

A three-dimensional homology model of lipid-free apolipoprotein A-IV using cross-linking and mass spectrometry.

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.

Modulation of apolipoprotein A-IV lipid binding by an interaction between the N and C termini.

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe(334), which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe(334) mutants. Here, we identify Trp(12) and Phe(15) as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic alpha-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.

The structure of apolipoprotein A-II in discoidal high density lipoproteins.

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.

Apolipoprotein structural organization in high density lipoproteins: belts, bundles, hinges and hairpins.

PURPOSE OF REVIEW: To summarize recent advances towards an understanding of the three-dimensional structures of the apolipoprotein components of HDL with a specific focus on high resolution models of apolipoprotein A-I. RECENT FINDINGS: Since the primary sequence was first reported, various models have been advanced for the structure of apolipoprotein A-I, the major protein constituent of HDL, in its lipid-free and lipid-bound forms. Unfortunately, the generation of experimental data capable of distinguishing among the competing models has lagged far behind. However, recent experimental strategies, including X-ray crystallography, applications of resonance energy transfer and mass spectrometry, have combined with sophisticated theoretical approaches to develop three-dimensional structural models of apolipoprotein A-I with previously unavailable resolution. SUMMARY: The recent synergy of sophisticated computer modeling techniques with hard experimental data has generated new models for apolipoprotein A-I in certain subclasses of HDL produced in vitro. The challenge now is to adapt and test these models in the more complex forms of HDL isolated directly from human plasma.

A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.

A mass spectrometric determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins.

Discoidal forms of high density lipoproteins (HDL) are critical intermediates between lipid-poor apolipoprotein A-I (apo A-I), the major protein constituent of HDL, and the mature spherical forms that comprise the bulk of circulating particles. Thus, many studies have focused on understanding apoA-I structure in discs reconstituted in vitro. Recent theoretical and experimental work supports a "belt" model for apoA-I in which repeating amphipathic helical domains run parallel to the plane of the lipid disc. However, disc-associated apoA-I can adopt several tertiary arrangements that are consistent with a belt orientation. To distinguish among these, we cross-linked near-neighbor Lys groups in homogeneous 96 A discs containing exactly two molecules of apoA-I. After delipidation and tryptic digestion, mass spectrometry was used to identify 9 intermolecular and 11 intramolecular cross-links. The cross-linking pattern strongly suggests a "double-belt" molecular arrangement for apoA-I in which two apoA-I molecules wrap around the lipid bilayer disc forming two stacked rings in an antiparallel orientation with helix 5 of each apoA-I in juxtaposition (LL5/5 orientation). The data also suggests the presence of an additional double-belt orientation with a shifted helical registry (LL5/2 orientation). Furthermore, a 78 A particle with two molecules of apoA-I fit a similar double-belt motif with evidence for conformational changes in the N-terminus and the region near helix 5. A comparison of this work to a previous study is suggestive that a third molecule of apoA-I can form a hairpin in larger particles containing three molecules of apoA-I.

Probing the effect of side chains on the conformation and stability of helical peptides via isotope-edited infrared spectroscopy.

The mechanism of helix stabilization or destabilization by different amino acids has been the subject of several experimental and theoretical studies; these studies suggest that large or bulky side chains may modulate helix stability by altering the hydration of the helix backbone. In this paper, we report a spectroscopic study to determine the effect of alanine to leucine substitutions on the conformation and solvation of specific segments of a model helical peptide. A 25-residue, alanine-rich, helical peptide [Ac-(AAAAK)(4)-AAAAY-NH(2) (AKA)] and its two leucine variants [Ac-LLLLK-(AAAAK)(3)-AAAAY-NH(2) (LKA) and Ac-(AAAAK)(4)-LLLLY-NH(2) (AKL)] were characterized by infrared (IR) and electronic circular dichroism (ECD) spectroscopies. Introduction of (13)C isotopes into specific, consecutive, backbone carbonyls for certain blocks of each of the peptides mentioned above allows the IR spectra to be interpreted in terms of the conformation and solvation of specific residues within the helix. These isotope-edited IR spectra of the leucine peptides do not show evidence of a decrease in the degree of backbone solvation compared to the alanines, but suggest that the peptide may adopt a distorted conformation to accommodate the larger leucine side chains at the N-terminus. These experiments demonstrate the power of isotope-edited IR in dissecting subtle changes in helix conformation at the residue level.

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study.

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.

Discrimination between peptide 3(10)- and alpha-helices. Theoretical analysis of the impact of alpha-methyl substitution on experimental spectra.

Detailed spectral simulations based on ab initio density functional theory computations of the amide I and II infrared (IR) and vibrational circular dichroism (VCD) spectra for Ac-(Ala)(4)-NH(2), Ac-(Aib-Ala)(2)-NH(2), and Ac-(Aib)(4)-NH(2) constrained to 3(10)- and alpha-helical conformations are presented. Parameters from these ab initio calculations are transferred onto corresponding larger oligopeptides to simulate the spectra for dodecamers. The differences between conformations and for different Aib substitution patterns within a conformation are reflected in observable spectral patterns where data are available. Simulated IR spectra show small frequency shifts in the amide I maxima between 3(10)- and alpha-helices, but the same magnitude shifts occur within one conformation upon Aib substitution. Thus, from a computational basis, the frequency of the amide I maximum does not discriminate between the 3(10)- or alpha-helical conformations. Calculated VCD band shapes for 3(10)-helices showed more significant changes in amplitude, with change in the fraction of Aib, than those for alpha-helices. Generally, with increasing Aib content, the overall amide I VCD intensity becomes weaker and the amide I couplet becomes more conservative, while the amide II VCD is less affected. Although the detailed band shape is shown to be sensitive to alpha-Me substitution, the basic pattern of amide I and II relative VCD intensities still differs between alpha- and 3(10)-helices and, as a consequence, successfully discriminates between them. These predictions are all borne out in experimental spectra of Aib, mixed Aib-Ala, and Ala-based helical peptides, where available.

Empirical relationships between isotope-edited IR spectra and helix geometry in model peptides.

Infrared spectroscopy (IR) is commonly used to study secondary structure of both peptides and proteins. The amide I band is very sensitive to peptide secondary structure, and the conformation of a peptide can be probed at the residue level by introducing site-specific isotope-labels into the peptide backbone. The replacement of a carbonyl (12)C with a (13)C results in a approximately 40 cm(-1) shift in the amide I' band. The amide I bands of specifically labeled helices should vary systematically as a function of the number and relative spacing of the labeled residues; thus one should be able to describe the conformation of a polypeptide in substantial detail by probing the changes in IR spectra as a function of the number and positioning of isotope labels. In this study, we report IR spectra of a series of differently labeled helical peptides. A series of 25mer peptides were synthesized based on the repeat sequence (AAAAK)(n). We have varied the number and spacing of the labels on each peptide and studied the changes in the (12)C and (13)C amide I' band due to label position. Our results indicate that changing the number of labels changes the frequency and intensity of both the (12)C and the (13)C amide mode. We also found that varying the spacing between labels causes these amide peaks to shift. Isotope labeling, combined with IR spectroscopy and theoretical predictions, may generate a description of peptide backbone conformations at the residue level.