Himalayan glaciers supply meltwater to densely populated catchments in South Asia, and regional observations of glacier change over multiple decades are needed to understand climate drivers and assess resulting impacts on glacier-fed rivers. Here, we quantify changes in ice thickness during the intervals 1975–2000 and 2000–2016 across the Himalayas, using a set of digital elevation models derived from cold war–era spy satellite film and modern stereo satellite imagery. We observe consistent ice loss along the entire 2000-km transect for both intervals and find a doubling of the average loss rate during 2000–2016 [−0.43 ± 0.14 m w.e. year−1 (meters of water equivalent per year)] compared to 1975–2000 (−0.22 ± 0.13 m w.e. year−1). The similar magnitude and acceleration of ice loss across the Himalayas suggests a regionally coherent climate forcing, consistent with atmospheric warming and associated energy fluxes as the dominant drivers of glacier change.

Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed. R ecently, ion channel research has been aided by the discovery of bacterial ion channels that share many key features of mammalian channels (1-3). Some of these bacterial channel proteins are smaller and more amenable to expression and purification than mammalian channels. Importantly, bacterial channels can often be reconstituted into vesicles and other synthetic membrane systems, allowing detailed functional characterization (4-6). Furthermore, several bacterial channels have been described at atomic resolution by x-ray crystallography (7-11), providing opportunities for correlating structure and function in a key class of membrane protein.Although the preparation of many medium-sized watersoluble proteins by chemical ligation is now a routine procedure (12)(13)(14), the synthesis of ion channels and other membrane proteins presents unique challenges. Many hydrophobic peptides have sequences that couple inefficiently, meaning that the synthesis of each segment must be optimized. Hydrophobic peptides also have limited solubility and tend to aggregate. Furthermore, after synthesis is complete the native oligomeric protein must be formed from unfolded monomers. However, recent advances in chemical ligation methodologies have permitted the semisynthesis (15) and the total chemical synthesis of membrane proteins, including the 97-residue type III (single membrane-spanning segment) M2 protein of the influenza virus (16). The synthetic M2 protein assembled into the native tetrameric form on reconstitution into dodecylphosphocholine micelles.Here, we describe the chemical synthesis of two polytopic membrane proteins, the mechanosensitive ion channels from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (TbMscL), and the vesicle reconstitution of these channels into a form functionally similar to that of the recombinant protein.Multimilligram quantities of Ec-MscL and Tb-MscL were generated by optimizing synthesis, purification, and ligation protocols previously developed fo...

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels.Since its cloning in 1994 by the Kung labs (1), the mechanosensitive channel of large conductance (MscL) 1 has developed into a prototype ion channel for understanding cellular mechanosensation (2-5). Much of what is known about the function of MscL has been gained through investigation of the Escherichia coli channel using electrophysiology and mutagenesis (3-5). Electrophysiological characterization of Ec-MscL in both bacterial spheroplasts and reconstituted lipid vesicles has demonstrated that MscL is opened by tension from the lipid bilayer (1), quantitated the tension required to open MscL (6), predicted the pore size of the open channel (7), and suggested that there are several discrete steps on the opening pathway (6). Mutagenesis studies also determined that the first transmembrane region of Ec-MscL lined the pore and established an occlusion of the channel in the vicinity of residue .A breakthrough in the study of MscL came with the report from the Rees labs (12) of the high resolution crystal structure of MscL from Mycobacterium tuberculosis (Tb-MscL). This result confirmed many of the essential conclusions of the earlier mutagenesis studies, while also clarifying some confusion concerning the stoichiometry of the channel and providing a wealth of new insights into the molecular details of the structure.The Interesting results have been obtained from molecular dynamics simulations beginning from the closed state of MscL (16 -18). At present, however, it is not possible to run such simulations long enough to see the transition from closed to open state. As a result, the de novo construction of molecular models for the open state and various intermediates on the opening pathway has been attempted.In particular, a detailed, atomic-level gating model for EcMscL has been developed by Sukharev, Guy, and coworkers (SG) (19 -21). Although the Rees crystal structure is of TbMscL, SG chose to model Ec-MscL so that use could be made of the much larger collection of experimental data that exist for this homologue. These data were used extensively in developing the computational model. In addition to emphasizing key residues identified from the mutagenesis stud...

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. The recent crystal structures of two bacterial ion channels, the KcsA potassium channel and the mechanosensitive channel MscL, provide unique opportunities to study ion channel structure-function relationships (1, 2). Concerning the MscL system, recent work has attempted to rationalize the extensive functional studies on Escherichia coli MscL (Eco-MscL) 1 (17). This difference may result from protein structural differences, a difference in interactions with lipids, or both.Sequence alignment is essential to map previously studied E. coli GOF mutations onto the M. tuberculosis MscL sequence. In this work we report an optimal sequence alignment of 35 MscL homologues and an analysis of regions of conservation and variability. Consistent with previous studies, we find greater conservation in the transmembrane regions than in the loop or intracellular regions. Interestingly, the various channels clearly fall into subfamilies based on sequence similarity, with Eco-MscL and Tb-MscL in different subfamilies.By using the optimal alignment, we have prepared Tb-MscL analogues of several critical Eco-MscL GOF mutations (Fig. 1A). Perhaps surprisingly, we find that several well established Eco-MscL GOF mutants do not translate to the Tb-MscL system. We also directly evaluate a predicted intersubunit hydrogen bond in the Tb-MscL crystal structure (Fig. 1B). Crosslinking studies establish that these residues are indeed close in the reconstituted channel and firmly establish the pentameric nature of the channel. Mutations of this pair generally lead to GOF mutants, suggesting an important functional role for this specific region of the channel. Interestingly, no analogous interaction is apparent in the Eco-MscL alignment. Our results indicate that the functional studies performed on the Eco-MscL channel may not map directly onto the Tb-MscL crystal structure. MATERIALS AND METHODSSequence Analysis-Multiple sequence alignments were obtained using alignment of multiple sequences (AMPS) (20,21), and consensus group analysis was performed using multiple EM for motif elicitation (MEME) (22,23). The alignment was broken into regions, extracellular