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Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 μs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.

Otosclerosis is a bone disorder of the otic capsule and common form of late-onset hearing impairment. Considered a complex disease, little is known about its pathogenesis. Over the past 20 years, ten autosomal dominant loci (OTSC1-10) have been mapped but no genes identified. Herein, we map a new OTSC locus to a 9.96 Mb region within the FOX gene cluster on 16q24.1 and identify a 15 bp coding deletion in Forkhead Box L1 co-segregating with otosclerosis in a Caucasian family. Phenotype ranges from moderate to severe hearing loss resolved by stapedectomy, to profound sensorineural loss requiring a cochlear implant. Mutant FOXL1 is both transcribed and translated and correctly locates to the cell nucleus. However, the deletion of 5 residues in the C-terminus of mutant FOXL1 causes a complete loss of transcriptional activity due to loss of secondary (alpha helix) structure. FOXL1 (rs764026385) was identified in a second unrelated case on a shared background. We conclude that FOXL1 (rs764026385) is pathogenic and causes autosomal dominant otosclerosis and propose a key inhibitory role for wildtype Foxl1 in bone remodelling in the otic capsule. New insights into the molecular pathology of otosclerosis from this study provide molecular targets for non-invasive therapeutic interventions.

While peptides can be excellent therapeutics for several conditions, their limited in vivo half-lives have been a major bottleneck in the development of therapeutic peptides. Conjugating the peptide to an inert chemical moiety is a strategy that has repeatedly proven to be successful in extending the half-life of some therapeutics. This systematic review and meta-analysis was conducted to examine the available literature and assess it in an unbiased manner to determine which conjugates, both biological and synthetic, provide the greatest increase in therapeutic peptide half-life. Systematic searches run on PubMed, Scopus and SciFinder databases resulted in 845 studies pertaining to the topic, 16 of these were included in this review after assessment against pre-specified inclusion criteria registered on PROSPERO (#CRD42020222579). The most common reasons for exclusion were non-IV administration and large peptide size. Of the 16 studies that were included, a diverse suite of conjugates that increased half-life from 0.1 h to 33.57 h was identified. Amongst these peptides, the largest increase in half-life was seen when conjugated with glycosaminoglycans. A meta-analysis of studies that contained fatty acid conjugates indicated that acylation contributed to a statistically significant extension of half-life. Additionally, another meta-analysis followed by a sensitivity analysis suggested that conjugation with specifically engineered recombinant peptides might contribute to a more efficient extension of peptide half-life as compared to PEGylation. Moreover, we confirmed that while polyethylene glycol is a good synthetic conjugate, its chain length likely has an impact on its effectiveness in extending half-life. Furthermore, we found that most animal studies do not include as much detail when reporting findings as compared to human studies. Inclusion of additional experimental detail on aspects such as independent assessment and randomization may be an easily accomplished strategy to drive more conjugated peptides towards clinical studies.

Much of the work probing antimicrobial peptide (AMP) mechanisms has focussed on how these molecules permeabilize lipid bilayers. However, AMPs must also traverse a variety of non-lipid cell envelope components before they reach the lipid bilayer. Additionally, there is a growing list of AMPs with non-lipid targets inside the cell. It is thus useful to extend the biophysical methods that have been traditionally applied to study AMP mechanisms in liposomes to the full bacteria, where the lipids are present along with the full complexity of the rest of the bacterium. This review focusses on what can be learned about AMP mechanisms from solid-state NMR of AMP-treated intact bacteria. It also touches on flow cytometry as a complementary method for measuring permeabilization of bacterial lipid membranes in whole bacteria.

Solid state NMR has been tremendously useful in characterizing the structure and dynamics of model membranes composed of simple lipid mixtures. Model lipid studies employing solid state NMR have included important work revealing how membrane bilayer structure and dynamics are affected by molecules such as antimicrobial peptides (AMPs). However, solid state NMR need not be applied only to model membranes, but can also be used with living, intact cells. NMR of whole cells holds promise for helping resolve some unsolved mysteries about how bacteria interact with AMPs. This mini-review will focus on recent studies using 2H NMR to study how treatment with AMPs affect membranes in intact bacteria.

The histidine-rich antimicrobial peptides Gad-1 and Gad-2, from paralogous genes in cod, provide an opportunity to examine the effect of charge and nonelectrostatic factors on peptide-vesicle interaction and on peptide antimicrobial activity. In this study, the dependence of vesicle ζ-potential on peptide concentration has been used to examine the binding of these peptides to model vesicle surfaces at pH = 5.0, for which the charges of Gad-1 and Gad-2 are +8 and +5, respectively, and at pH = 7.0, where their charges are +3 and +1, respectively. Interpreting the observed ζ-potential behaviors as examples of Langmuir adsorption isotherms, it is possible to infer the equilibrium constant for peptide-vesicle binding, the fraction of the peptide bound at low peptide concentration, and the maximum peptide-to-lipid ratio when the vesicle surface is saturated at high peptide concentration. For both peptides, higher peptide charge is found to be correlated with a lower fraction of the peptide being bound to vesicle surfaces at low peptide concentration and with a smaller maximum bound peptide-to-lipid ratio at high peptide concentration. The equilibrium binding constant, on the other hand, is more strongly correlated with the peptide sequence than with the charge. Gad-1, which has been shown to be more biologically active than Gad-2, displayed a significantly higher equilibrium binding constant. These observations suggest that while the maximum peptide density on the vesicle surface is limited by electrostatic interactions, the free energy of peptide binding, like the observed antimicrobial activities of the Gad peptides, is also sensitive to other peptide factors which might, for example, influence hydrophobic interactions.

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod. 15N and 2H solid state nuclear magnetic resonance (NMR) were used to characterize their lipid-bound structures and lipid interactions. Gad-1 was found to position in-plane in POPC: POPG bilayers. Gad-1 displayed greater effects than Gad-2 on lipid acyl chain order of POPE: POPG and POPE: POPG: CL bilayers, in keeping with its greater activity against E. coli. The effect of Gad-1 and Gad-2 on lipid bilayer order was only weakly affected by changes in pH, and hence changes in histidine charge. This was somewhat surprising for Gad-2 as this peptide's biological activity has been shown to be greater at low pH and thus the finding may point to the existence of functional interactions with non-lipid components of bacteria. The incorporation of cardiolipin into POPE: POPG bilayers in such a way as to preserve the overall charge of the bilayers did not alter Gad-1's effects on lipid acyl chain order parameters, which report on motions on the 10−5 s timescale. When cardiolipin and Gad-1 were both present, there were subtle changes on membrane dynamics at other timescales.

SP-B63–78, a lung surfactant protein fragment, and magainin 2, an antimicrobial peptide, are amphipathic peptides with the same overall charge but different biological functions. Deuterium nuclear magnetic resonance has been used to compare the interactions of these peptides with dispersions of 1,2-dimyristoyl-sn-glycero-3-phophocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phophocholine (DHPC) (4:1) and DMPC/1,2-dimyristoyl-sn-glycero-3-phopho-(1′-rac-glycerol) (DMPG)/DHPC (3:1:1), two mixtures of long-chain and short-chain lipids that display bicellar behavior. This study exploited the sensitivity of a bicellar system structural organization to factors that modify partitioning of their lipid components between different environments. In small bicelle particles formed at low temperatures, short-chain components preferentially occupy curved rim environments around bilayer disks of the long-chain components. Changes in chain order and lipid mixing, on heating, can drive transitions to more extended assemblies...

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B&rsquo, s structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B&rsquo, s structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B&rsquo, s &ldquo, central loop&rdquo, and &ldquo, hinge&rdquo, regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B&rsquo, s essential functions.

The lipid bilayer disrupting effect of antimicrobial peptides (AMPs) has been widely studied in model-lipid systems by applying biophysical techniques such as 2H NMR spectroscopy. Real bacteria cell envelopes contain non-lipid components, such as peptidoglycan, and thus it is important to assess the effects of such non-lipid components on the lipid-disrupting effects of AMPs. To this end, our group and other have developed methods that promote uptake of deuterium-labeled acyl chains in bacterial cells to produce 2H-membrane-enriched Bacillus subtilis. In this work, we studied changes in the static 2H NMR spectra of B. subtilis induced by the AMPs MSI-78 and BP100. Addition of both AMPs resulted in the increase of lipid acyl chain disorder consistent with disruption of the bacterial membrane. In addition, the peptide to lipid molar ratios (P:L) that give rise to observable effects fall between the P:L molar ratios necessary to generate membrane disruption in model-lipid-only systems and the P:L molar ratios needed to inhibit bacterial cell growth. This observation supports a role for the non-lipid components in modulating the AMP-lipid interactions.

Lung surfactant, besides alveolar stability, also provides defence against pathogens by surfactant proteins (SP), SP-A and SP-D. The hydrophobic proteins SP-B and SP-C enhance surface activity. An unusual and paradoxical effect of bovine LS and synthetic model LS with SP-B/-C was bactericidal to Staphylococcus aureus and Escherichia coli. Bacterial proliferation were investigated with bovine lung surfactant extract (BLES), dipalmitoylphosphatdylcholine, palmitooleylglycerol, in combination with SP-B/-C using standard microbiological colony forming unit (CFU) counts and structural imaging. BLES and other surfactant-SP-B/-C mixtures inhibit bacterial growth in the concentration range of 0 -7.5 mg/mL, at > 10 mg/mL paradoxical growth of both the bacterial species suggest antibiotic resistance. The lipid only LS have no effect on bacterial proliferation. Smaller peptide mimics of SP-B or SP-B1-25, were less efficient than SP-Cff. Ultra structural studies of the bacterial CFU using electron and atomic force microscopy suggest some membrane damage of S. aereus at inhibitory concentration of BLES, and some structural alteration of E. coli at dividing zones, suggesting utilization and incorporation of surfactant lipid species by both bacteria. The results depicted from in vitro studies are also in agreement with protein-protein interactions obtained from PatchDock, FireDock and ClasPro algorithm. The MD-simulation decipher a small range fluctuation of gyration radius of the LS proteins and their peptide mimics. The studies have alarming implications in the use of high dosages (100 mg/mL/kg body weight) of exogenous surfactant for treatment of respiratory distress syndrome, genetic knock-out abnormalities associated with these proteins, and the novel roles played by SP-B/C as bactericidal agents.

Human hepatic lipase (hHL) is a cell surface associated enzyme that hydrolyzes triacylglycerols and phospholipids within circulating lipoproteins. We hypothesized that an amino acid sequence mimicking the major heparin binding domain (HBD) of hHL will displace hHL from cell surfaces. To test this hypothesis, we generated a recombinant protein of thioredoxin linked with a cleavable, tagged sequence containing amino acids 442 to 476 of the mature hHL sequence, which contains the major HBD of hHL. The recombinant protein associated with heparin-sepharose, and its peak elution from heparin-sepharose occurred in the presence of 0.5 M NaCl. We cleaved and purified the tagged sequence containing the HBD from the recombinant protein and tested the ability of the peptide to displace full-length hHL from HEK-293 cells. The peptide indeed displaced hHL from cell surfaces, while no significant displacement was observed in the presence of a peptide with a scrambled sequence. Finally, we obtained structural information for the peptide containing the HBD. 1 H- and 15 N-NMR spectra of the peptide indicate the peptide is largely unstructured, although not completely random coil. The addition of heparin to the peptide induced some changes in chemical shift, suggesting changes in peptide structure and/or specific interactions with heparin. Molecular simulations confirm the largely unstructured nature of the isolated peptide, but they also indicate weak tendencies for both α- and β-structure formation in different parts of the chain. Overall, these data provide a proof-of-principle for the use of mimetic peptides for the displacement of cell surface associated lipases.

Discoveries relating to innate immunity and antimicrobial peptides (AMPs) granted Bruce Beutler and Jules Hoffmann a Nobel prize in medicine in 2011, and opened up new avenues for the development of therapies against infections, and even cancers. The mechanisms by which AMPs interact with, and ultimately disrupt, bacterial cell membranes is still, to a large extent, incompletely understood. Up until recently, this mechanism was studied using model lipid membranes that failed to reproduce the complexity of molecular interactions present in real cells comprising lipids but also membrane proteins, a cell wall containing peptidoglycan or lipopolysaccharides, and other molecules. In this review, we focus on recent attempts to study, at the molecular level, the interaction between cationic AMPs and intact bacteria, by

Gaduscidin-1 and -2 (GAD-1 and GAD-2) are antimicrobial peptides (AMPs) that contain several histidine residues and are thus expected to exhibit pH-dependent activity. In order to help elucidate their mechanism of membrane disruption, we have performed molecular dynamics simulations with the peptides in both histidine-charged and histidine-neutral forms, along with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid molecules. The simulations employed GROMACS software and an OPLS-AA force field. Initially, the peptide and lipids were placed randomly in the simulation box and then were allowed to self-assemble. The results demonstrated a marked preference for the regions of the peptides that contain sequential pairs of histidine residues to associate closely with bilayer pores. This preference is observed even when the histidines are in their uncharged form. It appears that the relative compactness and rigidity of histidine pairs require the more aqueous and disordered environment of the pores to satisfy hydrophilic interactions. The final peptide structures exhibited a wide variety of structures and topologies, with the most helical structures positioning most parallel to the bilayer surface and the less ordered structures interacting more closely with the pore. Thus, the results give atomistic insight into those models of AMP mechanism that promote the importance of structural heterogeneity and imperfect amphipathicity to AMP activity and selectivity.

Antimicrobial peptides (AMPs) may interact with a variety of target cell components, including the lipid bilayer, non-lipidic cell envelope components, and/or intracellular targets. However, most biophysical experiments aimed at elucidating the detailed mechanism of AMPs are limited to simple model membrane systems and neglect potentially functional interactions between AMPs and non-lipidic cell components. One of the biophysical techniques commonly used to study how AMPs interact with lipid bilayers is solid-state deuterium NMR. In this chapter we provide protocols to prepare deuterium-labeled intact Gram-negative and Gram-positive bacteria and to observe these samples using solid-state deuterium NMR. Such experiments have the potential to provide important information about how non-lipidic cell envelope components modulate AMP interactions with the cytoplasmic membrane of bacteria.

Lung surfactant protein B (SP-B), a 79 residue, hydrophobic protein from the saposin superfamily, plays an essential role in breathing. Because of the extreme hydrophobicity of SP-B, the experimental structure of this protein has not yet been determined. Here, we run all-atom molecular dynamics simulations using the OPLS-AA force field in GROMACS to study SP-B's structure and mechanisms for promoting lipid reorganization. Firstly, we find that the final structures indicate the need for some fine-tuning of the homology-based secondary structure predictions. Secondly, we find energetically feasible structures 1) with SP-B's helices in the plane of the bilayer, 2) with SP-B's helices inclined with respect to the bilayer, and 3) with SP-B in a closed structure interacting peripherally with the bilayer. Interestingly, SP-B structures that were bent at the hinge region between the pairs of helices promoted and/or stabilized defects in the lipid bilayer. Finally, particular salt bridge patterns and structural plasticity in the central loop and adjacent region of SP-B appeared to be involved in SP-B's lipid reorganization abilities.

The hydrophobic lung surfactant protein, SP-B, is essential for survival. Cycling of lung volume during respiration requires a surface-active lipid-protein layer at the alveolar air-water interface. SP-B may contribute to surfactant layer maintenance and renewal by facilitating contact and transfer between the surface layer and bilayer reservoirs of surfactant material. However, only small effects of SP-B on phospholipid orientational order in model systems have been reported. In this study, N-terminal (SP-B(8-25)) and C-terminal (SP-B(63-78)) helices of SP-B, either linked as Mini-B or unlinked but present in equal amounts, were incorporated into either model phospholipid mixtures or into bovine lipid extract surfactant in the form of vesicle dispersions or mechanically oriented bilayer samples. Deuterium and phosphorus nuclear magnetic resonance (NMR) were used to characterize effects of these peptides on phospholipid chain orientational order, headgroup orientation, and the response of lipid-peptide mixtures to mechanical orientation by mica plates. Only small effects on chain orientational order or headgroup orientation, in either vesicle or mechanically oriented samples, were seen. In mechanically constrained samples, however, Mini-B and its component helices did have specific effects on the propensity of lipid-peptide mixtures to form unoriented bilayer populations which do not exchange with the oriented fraction on the timescale of the NMR experiment. Modification of local bilayer orientation, even in the presence of mechanical constraint, may be relevant to the transfer of material from bilayer reservoirs to a flat surface-active layer, a process that likely requires contact facilitated by the formation of highly curved protrusions.

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8Å more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.

Antimicrobial peptides (AMPs) are a group of small peptides with antimicrobial effects against pathogens and have been well studied because of their promise to be part of the solution to the rising problem of antibiotic resistance. Biophysical studies with AMPs in model lipid systems are commonly used to study AMPs' permeabilizing effect on lipid bilayers. However, it is not clear if this membrane-permeabilizing characteristic is the only mechanism of cell killing. Studies suggest that at least some AMPs have additional targets, different from the lipid bilayer.

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of 2H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The 2H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the 2H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bac...

Surfactant protein A (SP-A) is the most abundant protein component of lung surfactant, a complex mixture of proteins and lipids. SP-A performs host defense activities and modulates the biophysical properties of surfactant in concerted action with surfactant protein B (SP-B). Current models of lung surfactant mechanism generally assume SP-A functions in its octadecameric form. However, one of the findings of this study is that when SP-A is bound to detergent and lipid micelles that mimic lung surfactant phospholipids, it exists predominantly as smaller oligomers, in sharp contrast to the much larger forms observed when alone in water. These investigations were carried out in sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG), and mixed LMPC + LMPG micelles, using solution and diffusion nuclear magnetic resonance (NMR) spectroscopy. We have also probed SP-A's interaction with Mini-B, a biologically active synthetic fragment of SP-B, in the presence of micelles. Despite variations in Mini-B's own interactions with micelles of different compositions, SP-A is found to interact with Mini-B in all micelle systems and perhaps to undergo a further structural rearrangement upon interacting with Mini-B. The degree of SP-A-Mini-B interaction appears to be dependent on the type of lipid headgroup and is likely mediated through the micelles, rather than direct binding.

Piscidins are a family of antimicrobial peptides (AMPs) from fish that constitute an important component of their innate immune system. Based on previously generated Atlantic cod (Gadus morhua) expressed sequence tags (ESTs), we identified sequences representing two paralogous AMP-like transcripts. These Atlantic cod paralogues were termed gaduscidins (GAD-1 and GAD-2), derived from the genus name Gadus. We obtained full-length cDNA sequences for these putative AMP-encoding transcripts using bi-directional rapid amplification of cDNA ends (RACE). GAD-1 and GAD-2 putative peptides exhibit sequence similarity with members of the piscidin family from teleost fish. Quantitative reverse transcription - polymerase chain reaction (QPCR) was utilized in transcript expression studies of GAD-1 and GAD-2. We examined the constitutive expression of these transcripts in six tissues (head kidney, blood, brain, gill, pyloric caecum, and spleen) of non-stressed juvenile cod; GAD-1 and GAD-2 transcripts were detected in all six tissues, with the highest expression of both transcripts being in spleen, head kidney, and gill. Transcript expression of GAD-1 and GAD-2 was also examined in immune tissues (spleen and head kidney) following intraperitoneal (IP) injection of formalin-killed, atypical Aeromonas salmonicida (ASAL) or phosphate-buffered saline (PBS control). Both transcripts were weakly (less than 4-fold) up-regulated by ASAL in spleen but non-responsive to ASAL in head kidney. Since GAD-1 and GAD-2 transcripts are highly expressed constitutively in immune-relevant tissues (e.g. spleen and head kidney), and are weakly induced in spleen following IP injection with bacterial antigens, they may represent important components of innate immunity in Atlantic cod.

These studies detail the altered structure-function relationships caused by oxidation of surfactant protein B (SP-B), a mode of damage thought to be important in acute respiratory distress syndrome (ARDS), a common and frequently fatal condition. An 18-residue fragment comprising the N-terminal helix of SP-B was investigated in oxidized and unmodified forms by solution and solid-state nuclear magnetic resonance (NMR), circular dichroism (CD), and molecular dynamics (MD) simulation. Taken together, the results indicate that tryptophan oxidation causes substantial disruptions in helical structure and lipid interactions. The structural modifications induced by tryptophan oxidation were severe, with a reduction in helical extent from approximately three helical turns to, at most, one turn, and were observed in a variety of solvent environments, including sodium dodecyl sulfate (SDS) micelles, dodecyl phosphocholine (DPC) micelles, and a 40% hexafluoro-2-propanol (HFIP) aqueous solution. The unmodified peptide takes on an orientation within lipid bilayers that is tilted approximately 30° away from an in-plane position. Tryptophan oxidation causes significant modifications to the peptide-lipid interactions, and the peptide likely shifts to a more in-plane orientation within the lipids. Interestingly, the character of the disruptions to peptide-lipid interactions caused by tryptophan oxidation was highly dependent on the charge of the lipid headgroup.

The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure-inducing conditions: 40% hexafluoro-2-propanol aqueous solution at 5 degrees C. Under these conditions it was possible to detect medium-range NOEs for the first time for this peptide. This allowed a much better-defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4-8 of Peptide T take on a well-defined structure with a heavy atom RMSD of 0.78 A. The structure is stabilized by hydrogen bonding to side-chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic gamma-turn.

Interleukin-8alpha (IL-8alpha) is an antimicrobial peptide derived from the chemokine IL-8. Solution NMR was used to determine the atomic-resolution structure of IL-8alpha in SDS micelles. Solid-state NMR and tryptophan fluorescence were used to probe the interaction of IL-8alpha with model membranes. The peptide interacted differently with anionic versus purely zwitterionic micelles or bilayers. Tryptophan fluorescence demonstrated a deeper position of Trp4 in SDS micelles and POPC/POPG bilayers compared to pure POPC bilayers, consistent with (2)H order parameters, which also indicated a deeper position of the peptide in POPC/POPG bilayers compared to POPC bilayers. Paramagnetic probe data showed that IL-8alpha was situated roughly parallel to the SDS micelle surface, with a slight tilt that positioned the N-terminus more deeply in the micelle compared to the C-terminus. (15)N solid-state NMR spectra indicated a similar, nearly parallel position for the peptide in POPC/POPG bilayers. (31)P and (2)H solid-state NMR demonstrated that the peptide did not induce the formation of any nonlamellar phases and did not significantly disrupt bilayer orientation in aligned model membranes composed of POPC or POPC and POPG.

Pulmonary surfactant provides for a lipid rich film at the lung air-water interface, which prevents alveolar collapse at the end of expiration. The films are likely enriched in the major surfactant component dipalmitoylphosphatidylcholine (DPPC), which, due to its saturated fatty acid chains, can withstand high surface pressures up to 70 mN/m, thereby reducing surface tension in that interface to very low values (close to 1 mN/m). Despite many experimental measurements in situ, as well as in vitro for native lung surfactant films, the exact mechanism by which other fluid lipid components of surfactant, in combination with surfactant proteins, allow for such low surface tension values to be reached is not well understood. We have performed molecular dynamics simulation of films composed of DPPC alone and in mixtures with other fluid and acidic lipid components of surfactant at the high densities relevant to the low surface tension regime. 10-50 ns simulations were performed with the software GROMACS, with 40-64 lipids molecules plus water, using 5 different lipid compositions and 7 different areas per lipid. The primary focus was to learn how differences in lipid composition affect the response of the monolayer to compression, such as the development of curvature or the loss of lipids to the exterior of the monolayer. The systems studied exhibit features of two of the major schools of thought of lung surfactant mechanisms, in that although unsaturated lipids did not appear to prevent the monolayers from achieving high surface pressure, POPG did appear to be selectively squeezed out of the DPPC/POPG monolayers at high lipid densities.

Surfactant protein B (SP-B) is essential for normal lung surfactant function, which is in itself essential to life. However, the molecular basis for SP-B's activity is not understood and a high-resolution structure for SP-B has not been determined. Mini-B is a 34-residue peptide with internal disulfide linkages that is composed of the N- and C-terminal helical regions of SP-B. It has been shown to retain similar activity to full-length SP-B in certain in vitro and in vivo studies. We have used solution NMR to determine the structure of Mini-B in the presence of micelles composed of the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, Mini-B forms two alpha-helices connected by an unstructured loop. Mini-B possesses a strikingly amphipathic surface with a large positively charged patch on one face of the peptide and a large hydrophobic patch on the opposite face. A tryptophan side chain extends outward from the peptide in a position to interact with lipids at the polar/apolar interface. Interhelix interactions are stabilized by both disulfide bonds and by interleaving of hydrophobic side chains from the two helices.

The main chain motional properties for a series of peptides that appear to have preferred conformations in solution have been systematically studied using solution-state nuclear magnetic resonance spectroscopy. The series of peptides were derived from the N-termini of pro-inflammatory chemokine proteins and HoxB1, a transcriptional regulator. As an unstructured control, a ten residue peptide was designed, synthesized, and found to be minimally structured from solution NMR data. The dynamic properties of the main chain for the peptides were assessed through longitudinal and transverse main chain 13Cα relaxation rates and the heteronuclear nuclear Overhauser effect. Motional parameters were interpreted using reduced spectral density mapping and compared with those derived from an extended Lipari–Szabo model in which the rotational correlation time was calculated for each main chain site of the peptide. Comparison of spectral density and Lipari–Szabo analyses for the peptides to those of the unstructured control peptide reveals significant differences in the dynamic behavior of the peptides. The amplitude of picosecond to nanosecond timescale motions for the main chain is observed to decrease for all of the chemokine peptides and HoxB1 over the regions that show partial structure at low temperatures. Comparatively, changes in picosecond to nanosecond timescale motions for the unstructured control peptide show no correlation with sequence position. These results indicate that there are distinguishable low temperature motional differences between an intrinsically unstructured peptide and peptides that have an inherent propensity to structure. Proteins 2007; © 2006 Wiley-Liss, Inc.

Human hepatic lipase (hHL) is bound to the cell surface via heparan sulfate proteoglycans (HPSGs) and hydrolyzes triglycerides and phospholipids within circulating lipoproteins. High-density lipoprotein (HDL) transports excess extrahepatic cholesterol to the liver for excretion. However, small lipid-deficient HDL generated by hHL action has the ability to accept more cholesterol. I hypothesized that a peptide mimicking the cell surface associations of hHL will displace hHL from cell surfaces, increasing its activity in cell culture media. To test this hypothesis, I have recombinantly expressed and purified a tagged mimetic peptide, encompassing the major heparin-binding domain of hHL. Results from in vitro lipase displacement assays showed that the peptide has an ability to displace active HPSG associated lipases. Data from proton nuclear magnetic resonance experiments showed that structural changes occur in the peptide in the presence of heparin. Overall, these data provide a foundation to use mimetic peptides for the displacement of cell surface associated lipases.

Antimicrobial polypeptides (AMPs) are an important component of the innate immune system of many different organisms and are generally amphipathic and cationic in nature. Electrostatic interactions between positively charged residues of the AMP and negatively charged lipids in pathogen membranes are an important component of AMP mechanism and specificity. AMPs such as Gad-1 and Gad-2 that are rich in histidine, an amino acid that is uncharged at neutral pH and positively charged at slightly acidic pH, can thus exhibit pH dependent activity. 2H solid state NMR spectroscopy was used to study the effect of Gad-1 and Gad-2 on model membranes at different pH values. The addition of peptides to the model membrane system POPE/POPG-d31 (3:1) results in a change in the splitting of spectrum both at pH 7 and pH 5. Further investigation of peptide effects on lipid acyl chain motion were determined through calculations of orientational order parameter values (SCD). The order parameter profile demonstrated that Gad-1 causes more lipid acyl chain disordering than Gad-2, at both pH 7 and pH 5. These results are consistent with Gad-1's greater activity in assays for bacterial growth inhibition. On the other hand, while Gad-2 is more active at inhibiting bacterial growth at low pH than at neutral pH, its effect on lipid acyl chain disorder is the same at pH 7 and pH 5.

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.

Complete and accurate NMR spectral assignment is a prerequisite for high-throughput automated structure determination of biological macromolecules. However, completely automated assignment procedures generally encounter difficulties for all but the most ideal data sets. Sources of these problems include difficulty in resolving correlations in crowded spectral regions, as well as complications arising from dynamics, such as weak or missing peaks, or atoms exhibiting more than one peak due to exchange phenomena. Smartnotebook is a semi-automated assignment software package designed to combine the best features of the automated and manual approaches. The software finds and displays potential connections between residues, while the spectroscopist makes decisions on which connection is correct, allowing rapid and robust assignment. In addition, smartnotebook helps the user fit chains of connected residues to the primary sequence of the protein by comparing the experimentally determined chemical shifts with expected shifts derived from a chemical shift database, while providing bookkeeping throughout the assignment procedure.

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.

Surfactant associated-protein B (SP-B) is the most essential protein component of lung surfactant; its absence is fatal. Lung surfactant associated proteins, especially SP-B, assist in the organization and rearrangement of the lipid monolayers and multilayers that coat the air-water interface of the alveolus, and so help to reduce the work of breathing and prevent lung collapse. Due to SP-B's unique hydrophobic nature however, the overall three dimensional structure of the protein is not yet determined, therefore the molecular basis for its activity is not clearly understood. Recently, our lab has managed to produce the protein in bacteria using recombinant DNA technology. Recombinant SP-B (rSP-B) is shown to retain the expected alpha helical conformation in different membrane mimetic conditions, over a range of temperatures as observed by far UV circular dichroism (CD) spectroscopy. Dynamic Light Scattering (DLS) of rSP-B suspended in detergent micelles indicates two populations, one with a hydrodynamic size of ∼3 nm and the other at ∼100 nm. Nanoparticle Tracking Analysis (NTA) confirmed the ∼100 nm species to be prominent. Preliminary 1D solution NMR experiments have also been carried out.Moreover, rSP-B also shows promising results in in vitro measures of functionality when tested with a Langmuir-Blodgett trough. Lipid films in the presence of rSP-B are able to promote multilayer formation when compressed to a sufficiently high surface pressure, comparable to that of clinical lung surfactants. What is more, films in the presence of rSP-B demonstrate superior compressibility and film recovery when compared to films compressed without rSP-B.

A key aspect of the antibacterial activity of membrane-active peptides and molecules is their interaction with lipid bilayers. Thus solid-state NMR (SS-NMR) studies of antimicrobial peptides (AMPs) in complex with model membrane bilayers have provided a wealth of valuable information regarding their mechanism of action. However, such simplified systems only approximate the lipid bilayers found in the much more complex cell envelopes that AMPs encounter when they are functioning in vivo. Microbe cell envelopes contain a number of additional components, such as lipopolysaccharides, peptidoglycans, membrane proteins, and complex lipid compositions, that likely affect how AMPs function. Consequently, there is a need to bridge the gap between the model membrane experiments we rely upon to reveal details of AMP mechanism and the complex environment AMPs actually encounter in vivo. We have thus developed novel approaches to accomplish 2H SS-NMR experiments with intact bacteria. By manipulating the growth conditions of either wild-type or mutated Escherichia coli, it is possible to achieve high levels of lipid deuteration in the bacteria and thus study phospholipid–AMP interactions within the context of intact bacteria. The novel techniques have been applied to the antimicrobial peptide MSI-78 and antimicrobial marennine pigments in order to reveal aspects of their mechanisms not addressable by other types of studies.

Protein W (gpW) from bacteriophage λ is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two α-helices and a single two-stranded β-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases. It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known. We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum . MtRBP5 contains a four-stranded β-sheet platform supporting two α-helices, one on each side of the β-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database. The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.