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Dive into the research topics where M. Mohammad is active.

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Featured researches published by M. Mohammad.


Journal of the American Chemical Society | 2008

Controlling a Single Protein in a Nanopore through Electrostatic Traps

Mohammad M. Mohammad; Sumit Prakash; and Andreas Matouschek; Liviu Movileanu

Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.


Nature Structural & Molecular Biology | 2007

Structural insight into OprD substrate specificity

Shyamasri Biswas; Mohammad M. Mohammad; Dimki R. Patel; Liviu Movileanu; Bert van den Berg

OprD proteins form a large family of substrate-specific outer-membrane channels in Gram-negative bacteria. We report here the X-ray crystal structure of OprD from Pseudomonas aeruginosa, which reveals a monomeric 18-stranded β-barrel characterized by a very narrow pore constriction, with a positively charged basic ladder on one side and an electronegative pocket on the other side. The location of highly conserved residues in OprD suggests that the structure represents the general architecture of OprD channels.


Journal of the American Chemical Society | 2012

Engineering a Rigid Protein Tunnel for Biomolecular Detection

Mohammad M. Mohammad; Raghuvaran Iyer; Khalil R. Howard; Mark P. McPike; Philip N. Borer; Liviu Movileanu

One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize, or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.


European Biophysics Journal | 2008

Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology

Mohammad M. Mohammad; Liviu Movileanu

Despite its fundamental and critical importance in molecular biology and practical medical biotechnology, how a polypeptide interacts with a transmembrane protein pore is not yet comprehensively understood. Here, we employed single-channel electrical recordings to reveal the interactions of short polypeptides and small folded proteins with a robust β-barrel protein pore. The short polypeptides were ∼25 residues in length, resembling positively charged targeting presequences involved in protein import. The proteins were consisted of positively charged pre-cytochrome b2 fragments (pb2) fused to the small ribonuclease barnase (∼110 residues, Ba). Single-molecule experiments exploring the interaction of a folded pb2-Ba protein with a single β-barrel pore, which contained negatively charged electrostatic traps, revealed the complexity of a network of intermolecular forces, including driving and electrostatic ones. In addition, the interaction was dependent on other factors, such as the hydrophobic content of the interacting polypeptide, the location of the electrostatic trap, the length of the pb2 presequence and temperature. This single-molecule approach together with protein design of either the interacting polypeptide or the pore lumen opens new opportunities for the exploration of the polypeptide–pore interaction at high temporal resolution. Such future studies are also expected to unravel the advantages and limitations of the nanopore technique for the detection and exploration of individual polypeptides.


Structure | 2008

Crystal Structure of the Outer Membrane Protein OpdK from Pseudomonas aeruginosa.

Shyamasri Biswas; Mohammad M. Mohammad; Liviu Movileanu; Bert van den Berg

In Gram-negative bacteria that do not have porins, most water-soluble and small molecules are taken up by substrate-specific channels belonging to the OprD family. We report here the X-ray crystal structure of OpdK, an OprD family member implicated in the uptake of vanillate and related small aromatic acids. The OpdK structure reveals a monomeric, 18-stranded beta barrel with a kidney-shaped central pore. The OpdK pore constriction is relatively wide for a substrate-specific channel (approximately 8 A diameter), and it is lined by a positively charged patch of arginine residues on one side and an electronegative pocket on the opposite side-features likely to be important for substrate selection. Single-channel electrical recordings of OpdK show binding of vanillate to the channel, and they suggest that OpdK forms labile trimers in the outer membrane. Comparison of the OpdK structure with that of Pseudomonas aeruginosa OprD provides the first qualitative insights into the different substrate specificities of these closely related channels.


Journal of Biological Chemistry | 2011

Redesign of a Plugged β-Barrel Membrane Protein

Mohammad M. Mohammad; Khalil R. Howard; Liviu Movileanu

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.


Journal of Physical Chemistry B | 2010

Impact of distant charge reversals within a robust β-barrel protein pore

Mohammad M. Mohammad; Liviu Movileanu

Among all beta-barrel pores, staphylococcal alpha-hemolysin (alphaHL), a heptameric transmembrane protein of known high-resolution crystal structure, features a high stability in planar lipid bilayers under a wide range of harsh experimental conditions. Here, we employed single-channel electrical recordings and standard protein engineering to explore the impact of two distant charge reversals within the interior of the beta-barrel part of the pore. The charge reversals were replacements of lysines with aspartic acids. A charge reversal within the structurally stiff region of the beta barrel near the pore constriction reduced the open-state current of the pore, but produced a quiet pore, showing current fluctuation-free channel behavior. In contrast, a charge reversal on the trans entrance, within the structurally flexible glycine-rich turn of the beta barrel, increased the open-state current and produced gating activity of the pore in the form of large-amplitude and frequent current fluctuations. Remarkably, cumulative insertion of the two distant charge reversals resulted in a large-amplitude permanent blockade of the beta barrel, as judged by both single-channel and macroscopic current measurements. The results from this work suggest that these distant charge reversals are energetically coupled, producing different impacts on the ionic transport, the unitary conductance and the open-state probability of the pore.


Integrative Biology | 2013

Determination of the quaternary structure of a bacterial ATP-binding cassette (ABC) transporter in living cells.

Deo R. Singh; Mohammad M. Mohammad; Suparna Patowary; Michael R. Stoneman; Julie A. Oliver; Liviu Movileanu; Valerică Raicu

Pseudomonas aeruginosa is a pathogenic Gram-negative bacterium that affects patients with cystic fibrosis and immunocompromised individuals. This bacterium coexpresses two unique forms of lipopolysaccharides (LPSs) on its surface, the A- and B-band LPS, which are among the main virulence factors that contribute to its pathogenicity. The polysaccharides in A-band LPSs are synthesized in the cytoplasm and translocated into the periplasm via an ATP-binding cassette (ABC) transporter consisting of a transmembrane protein, Wzm, and a cytoplasmic nucleotide-binding protein, Wzt. Most of the biochemical studies of A-band PSs in Pseudomonas aeruginosa are focused on the stages of the synthesis and ligation of PS, leaving the export stage involving the ABC transporter mostly unexplored. This difficulty is compounded by the fact that the subunit composition and structure of this bi-component ABC transporter are still unknown. Here we propose a simple but powerful method, based on Förster Resonance Energy Transfer (FRET) and optical micro-spectroscopy technology, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of the Wzm-Wzt complex in living cells. It is found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the structure and behavior of this ABC transporter will help develop antibiotics targeting the biosynthesis of the A-band LPS endotoxin.


Biophysical Journal | 2012

Inspection of the Engineered FhuA ΔC/Δ4L Protein Nanopore by Polymer Exclusion

David J. Niedzwiecki; Mohammad M. Mohammad; Liviu Movileanu

Extensive engineering of protein nanopores for biotechnological applications using native scaffolds requires further inspection of their internal geometry and size. Recently, we redesigned ferric hydroxamate uptake component A (FhuA), a 22-β-stranded protein containing an N-terminal 160-residue cork domain (C). The cork domain and four large extracellular loops (4L) were deleted to obtain an unusually stiff engineered FhuA ΔC/Δ4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of FhuA ΔC/Δ4L. When this nanopore was reconstituted into a synthetic planar lipid bilayer, addition of poly(ethylene glycols) produced modifications in the single-channel conductance, allowing for the evaluation of the nanopore diameter. Here, we report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance smaller than the trans entrance, in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Further experiments with impermeable dextran polymers indicated an average internal diameter of ~2.4 nm, a conclusion we arrived at based upon the polymer-induced alteration of the access resistance contribution to the nanopores total resistance. Molecular insights inferred from this work represent a platform for future protein engineering of FhuA that will be employed for specific tasks in biotechnological applications.


Biochimica et Biophysica Acta | 2016

Global redesign of a native β-barrel scaffold

Aaron J. Wolfe; Mohammad M. Mohammad; Avinash Kumar Thakur; Liviu Movileanu

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded β-barrel protein into a large-conductance protein pore. Here, we show that this redesigned β-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric β-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

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Deo R. Singh

University of Wisconsin–Milwaukee

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Julie A. Oliver

University of Wisconsin–Milwaukee

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