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1. Flanking aromatic residue competition influences transmembrane peptide helix dynamics

   https://doi.org/10.1002/1873-3468.13926  

   McKay, Matthew J. ; Greathouse, Denise V. ; Koeppe, II, Roger E. ( September 2020 , FEBS Letters)

   To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW^4,20ALP23 (acetyl‐GGAW⁴A(LA)₆LAW²⁰AGA‐amide) were examined using deuterium NMR spectroscopy. It was found that L^5,19GW^4,20ALP23, a sequence isomer of the low to moderately dynamic GW^5,19ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F^4,5GW²⁰ALP23 restores a low level of dynamic averaging, similar to that of the F^4,5GW¹⁹ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

    

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2. Protonation‐Driven Membrane Insertion of a pH‐Low Insertion Peptide

   https://doi.org/10.1002/ange.201605203  

   Hanz, Samuel Z. ; Shu, Nicolas S. ; Qian, Jieni ; Christman, Nathaniel ; Kranz, Patrick ; An, Ming ; Grewer, Christof ; Qiang, Wei ( August 2016 , Angewandte Chemie)

   The pH‐low insertion peptide (pHLIP) inserts into membranes and forms a transmembrane (TM) α‐helix in response to slight acidity, and has shown great potential for cancer diagnosis and treatment. As a lead, pHLIP is challenging to optimize because the mechanism of its pH‐dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change, the particular role played by each of them in the protonation‐driven insertion process is not clear. The precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid‐state NMR spectroscopy is used to address these central questions. Tracing backbone conformations revealed that the TM helix spans from A10 to D33 with a break at T19 to P20. Residue‐specific pK_avalues of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively, and define the sequence of protonations which lead to insertion. Furthermore, possible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.

    

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3. An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles

   https://doi.org/10.3390/v12121466  

   Varkey, Jobin ; Zhang, Jiantao ; Kim, Junghyun ; George, Gincy ; He, Guijuan ; Belov, George ; Langen, Ralf ; Wang, Xiaofeng ( December 2020 , Viruses)

   null (Ed.)

   Positive-strand RNA viruses universally remodel host intracellular membranes to form membrane-bound viral replication complexes, where viral offspring RNAs are synthesized. In the majority of cases, viral replication proteins are targeted to and play critical roles in the modulation of the designated organelle membranes. Many viral replication proteins do not have transmembrane domains, but contain single or multiple amphipathic alpha-helices. It has been conventionally recognized that these helices serve as an anchor for viral replication protein to be associated with membranes. We report here that a peptide representing the amphipathic α-helix at the N-terminus of the poliovirus 2C protein not only binds to liposomes, but also remodels spherical liposomes into tubules. The membrane remodeling ability of this amphipathic alpha-helix is similar to that recognized in other amphipathic alpha-helices from cellular proteins involved in membrane remodeling, such as BAR domain proteins. Mutations affecting the hydrophobic face of the amphipathic alpha-helix severely compromised membrane remodeling of vesicles with physiologically relevant phospholipid composition. These mutations also affected the ability of poliovirus to form plaques indicative of reduced viral replication, further underscoring the importance of membrane remodeling by the amphipathic alpha-helix in possible relation to the formation of viral replication complexes. 

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4. Factors That Control the Force Needed to Unfold a Membrane Protein in Silico Depend on the Mode of Denaturation

   https://doi.org/10.3390/ijms24032654  

   Faruk, Nabil F. ; Peng, Xiangda ; Sosnick, Tobin R. ( February 2023 , International Journal of Molecular Sciences)

   Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane–protein interactions had only moderate influences, except for the extraction of the last helix where the membrane–protein interactions had a stronger influence. The up–down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein. 

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5. Determine both the conformation and orientation of a specific residue in α-synuclein(61–95) even in monolayer by 13C isotopic label and p-polarized multiple-angle incidence resolution spectrometry (pMAIRS)

   https://doi.org/10.1007/s44211-022-00128-0  

   Wang, Chengshan ; Zhou, Yiqun ; Ewuola, Christopher ; Akinleye, Toyin ; Hasegawa, Takeshi ; Leblanc, Roger M. ( May 2022 , Analytical Sciences)

   Protein’s magic function stems from its structure and various analytical techniques have been developed for it. Among proteins, membrane proteins are encoded 20–30% of genomes, whereas cause challenges for many analytical techniques. For example, lots of membrane proteins cannot form single crystal structure required by X-ray crystallography. As for NMR, the measurements were hindered by the low tumbling rates of membrane (i.e., phospholipid bilayers) where membrane proteins exist. In addition, membrane proteins usually lay parallel to the surface of phospholipid bilayers or form transmembrane structure. No matter parallel or perpendicular to phospholipid bilayers surface, membrane proteins form monolayer structure which is also difficult for X-ray and NMR to provide high-resolution results. Because NMR and X-ray crystallography are the two major analytical techniques to address protein’s structure, membrane proteins only contribute 2.4% to the solved protein databank. Surface FT-IR techniques can evaluate the conformation and orientation of membrane proteins by amide I band. Specifically for α-helical peptides/proteins, the orientation of the axis is critical to decide whether proteins form transmembrane structure. Notice that the traditional FT-IR can only provide “low-resolution” results.Here,¹³C isotope was introduced into the nonamyloid component (NAC), which spans residues 61–95 of α-synuclein (α-syn). Then, p-polarized multiple-angle incidence resolution spectrometry (pMAIRS) was used to determine the orientation of a specific residue of α-helical NAC in monolayer. In general, pMAIRS is a novel technique to work complementary with X-ray and NMR to address membrane peptides/proteins structure with high resolution even in monolayer.

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