Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo

Gas vesicles are gas‐filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid‐state NMR results suggest a coil‐α‐β‐β‐α‐coil fold. We obtained a first structural model of GvpA by high‐performance de novo modelling. Attenuated total reflection (ATR)‐Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti‐parallel β‐strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α‐helix I and α‐helix II, but also in the β‐turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.

[1]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[2]  F. Pfeifer,et al.  A p-loop motif and two basic regions in the regulatory protein GvpD are important for the repression of gas vesicle formation in the archaeon Haloferax mediterranei. , 2001, Microbiology.

[3]  E. Goormaghtigh,et al.  Evaluation of the information content in infrared spectra for protein secondary structure determination. , 2006, Biophysical journal.

[4]  Arne Elofsson,et al.  3D-Jury: A Simple Approach to Improve Protein Structure Predictions , 2003, Bioinform..

[5]  Kay Hamacher,et al.  BioPhysConnectoR: Connecting Sequence Information and Biophysical Models , 2010, BMC Bioinformatics.

[6]  K. Diederichs,et al.  Crystallization and preliminary X-ray crystallographic studies of the native and chemically modified anion-selective porin from Comamonas acidovorans. , 1998, Acta crystallographica. Section D, Biological crystallography.

[7]  C. Englert,et al.  Three different but related gene clusters encoding gas vesicles in halophilic archaea. , 1992, Journal of molecular biology.

[8]  Richard Bonneau,et al.  An improved protein decoy set for testing energy functions for protein structure prediction , 2003, Proteins.

[9]  K. Schmitz,et al.  GvpE- and GvpD-mediated transcription regulation of the p-gvp genes encoding gas vesicles in Halobacterium salinarum. , 2004, Microbiology.

[10]  A. Walsby,et al.  Crystalline structure of the gas vesicle wall from Anabaena flos-aquae. , 1976, Journal of molecular biology.

[11]  A. Schug,et al.  Basin hopping simulations for all-atom protein folding. , 2006, The Journal of chemical physics.

[12]  R. Griffin,et al.  Solid-state NMR evidence for inequivalent GvpA subunits in gas vesicles. , 2009, Journal of molecular biology.

[13]  Arne Elofsson,et al.  MaxSub: an automated measure for the assessment of protein structure prediction quality , 2000, Bioinform..

[14]  A. Walsby,et al.  Gas vesicles , 1994, Microbiological reviews.

[15]  F. Pfeifer,et al.  Anaerobiosis inhibits gas vesicle formation in halophilic Archaea , 2009, Molecular microbiology.

[16]  David Baker,et al.  Protein-protein docking with backbone flexibility. , 2007, Journal of molecular biology.

[17]  R. Jernigan,et al.  Anisotropy of fluctuation dynamics of proteins with an elastic network model. , 2001, Biophysical journal.

[18]  S. DasSarma,et al.  Complexity of Gas Vesicle Biogenesis in Halobacterium sp. Strain NRC-1: Identification of Five New Proteins , 2004, Journal of bacteriology.

[19]  C. Englert,et al.  Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac‐region boundary and suggests a regulatory role for the gvpD gene or its product , 1992, Molecular microbiology.

[20]  J A McCammon,et al.  Computing the Amino Acid Specificity of Fluctuations in Biomolecular Systems. , 2006, Journal of chemical theory and computation.

[21]  Wolfgang Wenzel,et al.  Template‐free protein structure prediction and quality assessment with an all‐atom free‐energy model , 2009, Proteins.

[22]  Kevin Karplus,et al.  PREDICT-2ND: a tool for generalized protein local structure prediction , 2008, Bioinform..

[23]  M. Fändrich,et al.  FTIR reveals structural differences between native β‐sheet proteins and amyloid fibrils , 2004, Protein science : a publication of the Protein Society.

[24]  Wolfgang Wenzel,et al.  A free-energy approach for all-atom protein simulation. , 2009, Biophysical journal.

[25]  H. Mantsch,et al.  The use and misuse of FTIR spectroscopy in the determination of protein structure. , 1995, Critical reviews in biochemistry and molecular biology.

[26]  F. Pfeifer,et al.  Regulation of gvp genes encoding gas vesicle proteins in halophilic Archaea , 2008, Archives of Microbiology.

[27]  R. Huber,et al.  Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. , 1995, Science.

[28]  Jonathan Casper,et al.  Combining local‐structure, fold‐recognition, and new fold methods for protein structure prediction , 2003, Proteins.

[29]  A. Blaurock,et al.  Structure of the wall of Halobacterium halobium gas vesicles. , 1976, Journal of molecular biology.

[30]  Frederick P. Brooks,et al.  Computing smooth molecular surfaces , 1994, IEEE Computer Graphics and Applications.

[31]  David Baker,et al.  Protein Structure Prediction Using Rosetta , 2004, Numerical Computer Methods, Part D.

[32]  W. Baumeister,et al.  Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli , 1992, FEBS letters.

[33]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[34]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[35]  J. Herzfeld,et al.  Subunit structure of gas vesicles: a MALDI-TOF mass spectrometry study. , 2004, Biophysical journal.

[36]  M. Miles,et al.  Direct observation of protein secondary structure in gas vesicles by atomic force microscopy. , 1996, Biophysical journal.

[37]  Richard Bonneau,et al.  De novo prediction of three-dimensional structures for major protein families. , 2002, Journal of molecular biology.

[38]  J. Walker,et al.  Complete amino acid sequence of cyanobacterial gas-vesicle protein indicates a 70-residue molecule that corresponds in size to the crystallographic unit cell. , 1986, The Biochemical journal.

[39]  Frederick P. Brooks,et al.  Linearly Scalable Computation of Smooth Molecular Surfaces , 1997 .

[40]  F. Pfeifer,et al.  Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements , 1993, Molecular and General Genetics MGG.

[41]  S. Offner,et al.  Eight of Fourteen gvp Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea , 2000, Journal of bacteriology.

[42]  Yang Zhang,et al.  I-TASSER server for protein 3D structure prediction , 2008, BMC Bioinformatics.

[43]  K. Hamacher,et al.  Relating sequence evolution of HIV1-protease to its underlying molecular mechanics. , 2008, Gene.

[44]  Liam J. McGuffin,et al.  Protein structure prediction servers at University College London , 2005, Nucleic Acids Res..

[45]  S. J. Beard,et al.  The sequence of the major gas vesicle protein, GvpA, influences the width and strength of halobacterial gas vesicles. , 2002, FEMS microbiology letters.

[46]  R. Griffin,et al.  Solid-state NMR characterization of gas vesicle structure. , 2010, Biophysical journal.

[47]  W. Stoeckenius,et al.  FURTHER CHARACTERIZATION OF PARTICULATE FRACTIONS FROM LYSED CELL ENVELOPES OF HALOBACTERIUM HALOBIUM AND ISOLATION OF GAS VACUOLE MEMBRANES , 1968, The Journal of cell biology.

[48]  M. Sternberg,et al.  Protein structure prediction on the Web: a case study using the Phyre server , 2009, Nature Protocols.

[49]  C. Englert,et al.  Analysis of gas vesicle gene expression in Haloferax mediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins. , 1993, The Journal of biological chemistry.

[50]  T L Blundell,et al.  FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. , 2001, Journal of molecular biology.

[51]  Gerrit Groenhof,et al.  GROMACS: Fast, flexible, and free , 2005, J. Comput. Chem..