Peptide Insertions

Typically, the insertion induces sharp variation of the membrane profile at the distances 0.5-1.0nm from the membrane-peptide interface [79-82]. The steepness of this perturbation indicates that the short-A, behavior of membrane moduli must be important in the estimates of the elastic energy. In addition, a peptide inserted in a membrane almost certainly perturbs the membrane s elastic moduli in the immediate vicinity of the inclusion. Both these effects, membrane nonlocality and nonuniform modification of elastic properties by insertions, might play an important role in resolving the contradiction between the local calculations [80] and the experimental data for the mean lifetime of a gramicidin channel [81,109,110]. ... 

The association of host defense peptides with lipid bilayers has been observed to be directly related to the ratio of peptide to lipid. At low peptide/lipid ratios, peptides are oriented parallel to the membrane. As the ratio increases, the peptides reorient themselves perpendicular to the membrane, ultimately inserting into the bilayer. Following membrane insertion transmembrane pores are formed. The insertion of peptides into the lipid membrane and subsequent translocation of peptides into the cytoplasm or formation of transmembrane pores has been described by multiple models of host defense peptide insertion. 

Warrington, K. H., Gorbatyuk, O. S., Harrison, J. K. et al. (2004). Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J. Virol. 78(12), 6595-6609. 

Because of the long time scales involved, it is currently not possible to simulate the process of peptide insertion at full atomic level description. As a consequence, in MD simulations a configuration is given, either parallel to the bilayer or inserted into it, and the evolution of the system is followed. Another approach is to perform simulations of orientational preference with the mean field approximation of lipids and water, retaining an atomic level description of the peptide. Several such simulations have been performed to determine the most likely mechanism of action for a given antimicrobial peptide. A recent review on simulations performed with antimicrobial peptides is given in ref. 78. 

At the time of writing this chapter, three commercial random peptide display libraries are available. Two phage Ph.D. libraries (New England Biolabs) are constituted of random 7-mers and random 12-mers fused to pill protein of the filamentous phage Ml3. The FliTrx (Invitrogen) is an E. coli display library with constrained random dodeca-peptides inserted into flagellin-thioredoxin fusion protein [10]. 

Kise Y, Lee SW, Park SG, Fukai S, Sengoku T, Ishii R, Yokoyama S, Kim S, Nureki O. A short peptide insertion crucial for an-giostatic activity of human tryptophanyl-tRNA synthetase. Nat. Struct. Mol. Biol. 2004 11 149-156. 

Shaw JE, Epand RE, Sinnathamby K, Li Z, Bittman R, Epand RM, Yip CM. Tracking peptide-membrane interactions insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly. J. Struct. Biol. 2006 155 458-469. 

NH2-temiinus resulting in a mass of about 30 IdD and 3) an external peptide standard (450 pmol) that was provided dry in an eppendoiftube and that had the same amino acid composition as the unique tryptic peptide insert but whose sequence was randomized. In the case of the two protein samples, 70 pmol of each had been subjected to SDS PAGE and were supplied either as Coomassie Blue stained gel slices or as a section of amido black stained PVDF membrane. In the case of the PVDF samples, an oversize piece of PVDF was included so that a section could be used as a digest control, and in the case of the gel samples, a blank section of gel was included for the same purpose in a separate eppendorf tube. 

The primary concerns in choosing the sequence of the unique, peptide insert were that a tryptic digestion of the 30 kD sample would release the target... 

A synthetic peptide analogue of the unique insert actually eluted at about 28% CHjCN both from a Vydac C-18 column on the system being tested (data not shown) and from a Zorbax C-18 column on an HPLC system located in a different laboratory (Fig. 1). In the latter instance the peptide insert eluted close to a minor peak eluting at about 54 min in the 28 kD chromatogram. As noted previously, the external peptide standard had the same amino acid composition but a different sequence from that of the unique peptide insert. 

Step 2. The signal peptide inserts into the membrane. We propose that the polar c region of the signal sequence resides transiently in the aqueous region of the periplasmic or lumenal side of the membrane. 

Valincius et al. used NR and ex situ electrochemical impedance spectroscopy to study the effect of amyloid (J-pephde insertion on supported hpid bilayers [60]. It has been speculated that the interaction between these oligomeric proteins and bilayer membranes plays an important role in the aggregation and protein misfold-ing that leads to various neurodegenerahve diseases including Alzehimer s and Parkinson s diseases [61]. Using a variety of solvent and phospholipid contrasts, including the use of perdeuterated phosphohpids, NR demonstrated that amyloid P-peptides inserted into hpid bilayers tethered to a gold-modified silicon substrate. 

Introduction of a peptide into the AAV2 capsid is a viable strategy for retargeting AAV2 to additional cell types however, modifications in the AAV2 capsid often result in a reduction in vector yields, especially if the insertion is large. The unique N-terminal region of VP2 allows for peptide insertions without a loss of titer, when... 

Pick 10-20 well separated colonies from the titer results and sequence the random peptide insert (see Notes 23 and 24). 

Phage liberty with 10 random peptide inserts... 

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