Investigation of the juxtamembrane region of neuronal-Synaptobrevin in synaptic transmission at theDrosophilaneuromuscular junction

In this study, the juxtamembrane region of the Drosophila SNARE protein neuronal-Synaptobrevin (n-Syb) was tested for its role in synaptic transmission. A transgenic approach was used to express n-Syb mutant genes. The transgenes carried engineered point mutations that alter the amino acid sequence of the conserved tryptophan residues in the juxtamembrane sequence. Such transgenes were expressed in an n-syb hypomorphic background, which produces little endogenous protein. On their own, hypomorphic flies displayed severe motor inhibition, limited life span, reduced evoked junctional potentials (EJPs), decreased synchronicity in EJP time to peak, and potentiation of EJPs with 10-Hz stimulation. All of these deficits were restored to wild-type levels with the expression of wild-type transgenic n-syb, regulated by the endogenous promoter ( n-sybWT). We created transgenic mutants with one additional tryptophan ( n-sybWW) or one less tryptophan ( n-sybAA) than the wild-type sequence. While n-sybWWresembled n-sybWTin all variables listed, n-sybAAexhibited decreased EJP amplitude, synchronicity, and quantal content. To determine whether the n-syb juxtamembrane region is important for transduction of force arising from SNARE complex assembly during membrane fusion, we introduced short 6-amino acid ( n-sybL6) or long 24-amino acid ( n-sybL24) flexible linkers into the n-syb transgene. We observed a reduced EJP amplitude in n-sybL6but not n-sybL24, while both linker mutants showed a decreased quantal content and an effect on the readily releasable and recycling vesicle pools. In conclusion, mutation of the juxtamembrane region of n-syb deleteriously affected synaptic transmission at the Drosophila neuromuscular junction.

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Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1406-1414.1989 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1406-1414

Author(s):

A A McCracken ◽

K B Kruse ◽

J L Brown

Keyword(s):

Amino Acid ◽

Proteinase Inhibitor ◽

Conformational Stability ◽

Point Mutations ◽

Salt Bridge ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Functional Importance ◽

Bridge Formation ◽

Cos Cells

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

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Detection of Point Mutations in rpoBGene of Rifampin-Resistant Rickettsia typhi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1845 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1845-1846 ◽

Cited By ~ 13

Author(s):

Jill Michelle Troyer ◽

Suzana Radulovic ◽

Siv G. E. Andersson ◽

Abdu F. Azad

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Wild Type ◽

Nucleotide Substitutions ◽

Rickettsia Typhi ◽

Rifampin Resistance

ABSTRACT The rpoB gene of rifampin-resistant Rickettsia typhi (Rif mutant) and wild-type R. typhi were sequenced and compared. The Rif mutant rpoB had three nucleotide substitutions, which resulted in amino acid changes at residues 151, 201, and 271 and may be the basis for the rifampin resistance.

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Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1406 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1406-1414 ◽

Cited By ~ 51

Author(s):

A A McCracken ◽

K B Kruse ◽

J L Brown

Keyword(s):

Amino Acid ◽

Proteinase Inhibitor ◽

Conformational Stability ◽

Point Mutations ◽

Salt Bridge ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Functional Importance ◽

Bridge Formation ◽

Cos Cells

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Calcium Binding Is Required for Calmodulin Function in Aspergillus nidulans

Eukaryotic Cell ◽

10.1128/ec.01.1.119-125.2002 ◽

2002 ◽

Vol 1 (1) ◽

pp. 119-125 ◽

Cited By ~ 18

Author(s):

James D. Joseph ◽

Anthony R. Means

Keyword(s):

Amino Acid ◽

Aspergillus Nidulans ◽

Calcium Binding ◽

Structural Basis ◽

Wild Type ◽

Endogenous Protein ◽

Chimeric Molecules ◽

Overlay Assay

ABSTRACT To explore the structural basis for the essential role of calmodulin (CaM) in Aspergillus nidulans, we have compared the biochemical and in vivo properties of A. nidulans CaM (AnCaM) with those of heterologous CaMs. Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca2+ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB. In contrast, although vertebrate CaM (VCaM) binds a spectrum of proteins similar to that for AnCaM, it is unable to fully activate CMKA and CMKB, displaying a higher K CaM and reduced V max for both enzymes. In correlation with the biochemical analysis, neither ScCaM nor 1234 can support A. nidulans growth in the absence of the endogenous protein, whereas VCaM only partially complements the absence of wild-type CaM. Analysis of VCaM and AnCaM chimeras demonstrates that amino acid variations in both N- and C-terminal domains contribute to the inability of VCaM to activate CMKB, but differences in the N terminus are largely responsible for the reduced activity towards CMKA. In vivo, the chimeric molecules support growth equivalently, but only to levels intermediate between those of VCaM and AnCaM, suggesting that the reduced ability to activate the CaMKs is not solely responsible for the inability of VCaM to complement the absence of the wild-type protein. Thus, not only is Ca2+ binding required for CaM function in A. nidulans, but the essential in vivo functions of A. nidulans CaM are uniquely sensitive to the subtle amino acid variations present in vertebrate CaM.

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CXCR4 and CCR5 Genetic Polymorphisms in Long-Term Nonprogressive Human Immunodeficiency Virus Infection: Lack of Association with Mutations other than CCR5-Δ32

Journal of Virology ◽

10.1128/jvi.72.7.6215-6217.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 6215-6217 ◽

Cited By ~ 43

Author(s):

Oren J. Cohen ◽

Stefania Paolucci ◽

Steven M. Bende ◽

MaryBeth Daucher ◽

Hiroyuki Moriuchi ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Point Mutations ◽

Predicted Amino Acid Sequence ◽

Wild Type ◽

Coding Sequences ◽

Coding Sequence ◽

Immunodeficiency Virus ◽

Hiv Envelope

ABSTRACT Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Δ32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Δ32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.

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Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility inEnterococcus faecalis

mBio ◽

10.1128/mbio.00361-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 12

Author(s):

Louis B. Rice ◽

Charlene Desbonnet ◽

Amelia Tait-Kamradt ◽

Monica Garcia-Solache ◽

John Lonks ◽

...

Keyword(s):

Amino Acid ◽

Enterococcus Faecalis ◽

Active Site ◽

Fluorescent Protein ◽

Joint Infection ◽

Point Mutations ◽

Promoter Sequence ◽

Amino Acid Substitutions ◽

Wild Type ◽

Content Type

ABSTRACTEnterococcus faecalisstrains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report anE. faecalisstrain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yieldedE. faecaliswith MICs of penicillins and carbapenems higher than those for wild-type strainE. faecalisJH2-2. Sequence analysis of thepbp4gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predictedpbp4−35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription ofpbp4in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed inE. faecalisLS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalisis an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report anE. faecalisstrain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

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The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

Infection and Immunity ◽

10.1128/iai.00135-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5595-5601 ◽

Cited By ~ 10

Author(s):

Cynthia L. Sears ◽

Simy L. Buckwold ◽

Jai W. Shin ◽

Augusto A. Franco

Keyword(s):

Biological Activity ◽

Amino Acid ◽

Point Mutations ◽

Bacteroides Fragilis ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Reverse Transcription Pcr ◽

C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

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Packpred: Predicting the functional effect of missense mutations

10.1101/2020.12.30.424909 ◽

2021 ◽

Author(s):

Kuan Pern Tan ◽

Tejashree Rajaram Kanitkar ◽

Kwoh Chee Keong ◽

M.S. Madhusudhan

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Substitution Matrix ◽

Saturation Mutagenesis ◽

Data Sets ◽

Missense Mutations ◽

Wild Type ◽

Data Set ◽

Functional Consequences

1.AbstractPredicting the functional consequences of single point mutations has relevance to protein function annotation and to clinical analysis/diagnosis. We developed and tested Packpred that makes use of a multi-body clique statistical potential in combination with a depth dependent amino acid substitution matrix (FADHM) and positional Shannon Entropy to predict the functional consequences of point mutations in proteins. Parameters were trained over a saturation mutagenesis data set of T4-lysozyme (1966 mutations). The method was tested over another saturation mutagenesis data set (CcdB; 1534 mutations) and the Missense3D data set (4099 mutations). The performance of Packpred was compared against those of six other contemporary methods. With MCC values of 0.42, 0.47 and 0.36 on the training and testing data sets respectively, Packpred outperforms all method in all data sets, with the exception of marginally underperforming to FADHM in the CcdB data set. On analyzing the results, we could build meta servers that chose best performing methods of wild type amino acids and for wild type-mutant amino acid pairs. This lead to an increase of MCC value of 0.40 and 0.51 for the two meta predictors respectively on the Missense3D data set. We conjecture that it is possible to improve accuracy with better meta predictors as among the 7 methods compared, at the least one method or another is able to correctly predict ∼99% of the data.

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Packpred: Predicting the Functional Effect of Missense Mutations

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.646288 ◽

2021 ◽

Vol 8 ◽

Author(s):

Kuan Pern Tan ◽

Tejashree Rajaram Kanitkar ◽

Chee Keong Kwoh ◽

Mallur Srivatsan Madhusudhan

Keyword(s):

Amino Acid ◽

Single Point ◽

Point Mutations ◽

Substitution Matrix ◽

Saturation Mutagenesis ◽

Data Sets ◽

Missense Mutations ◽

Wild Type ◽

Data Set ◽

Functional Consequences

Predicting the functional consequences of single point mutations has relevance to protein function annotation and to clinical analysis/diagnosis. We developed and tested Packpred that makes use of a multi-body clique statistical potential in combination with a depth-dependent amino acid substitution matrix (FADHM) and positional Shannon entropy to predict the functional consequences of point mutations in proteins. Parameters were trained over a saturation mutagenesis data set of T4-lysozyme (1,966 mutations). The method was tested over another saturation mutagenesis data set (CcdB; 1,534 mutations) and the Missense3D data set (4,099 mutations). The performance of Packpred was compared against those of six other contemporary methods. With MCC values of 0.42, 0.47, and 0.36 on the training and testing data sets, respectively, Packpred outperforms all methods in all data sets, with the exception of marginally underperforming in comparison to FADHM in the CcdB data set. A meta server analysis was performed that chose best performing methods of wild-type amino acids and for wild-type mutant amino acid pairs. This led to an increase in the MCC value of 0.40 and 0.51 for the two meta predictors, respectively, on the Missense3D data set. We conjecture that it is possible to improve accuracy with better meta predictors as among the seven methods compared, at least one method or another is able to correctly predict ∼99% of the data.

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Extracellular Loops of Lipid A 3-O-Deacylase PagL Are Involved in Recognition of Aminoarabinose-Based Membrane Modifications in Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.00587-08 ◽

2008 ◽

Vol 190 (16) ◽

pp. 5597-5606 ◽

Cited By ~ 15

Author(s):

Takayuki Manabe ◽

Kiyoshi Kawasaki

Keyword(s):

Amino Acid ◽

Salmonella Enterica ◽

Lipid A ◽

Salmonella Enterica Serovar Typhimurium ◽

Point Mutations ◽

Amino Acid Residues ◽

Wild Type ◽

Extracellular Loops ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium modifies its lipopolysaccharide (LPS), including the lipid A portion, in response to changes in its environment including host tissues. The lipid A 3-O-deacylase PagL, the expression of which is promoted under a host-mimetic environment, exhibits latency in S. enterica; deacylation of lipid A is not usually observed in vivo, despite the expression of the outer membrane protein PagL. In contrast, PagL does not exhibit latency in S. enterica pmrA and pmrE mutants, both of which are deficient in the aminoarabinose-based modification of lipid A, indicating that aminoarabinose-modified LPS species were involved in the latency. In order to analyze the machinery for PagL's repression, we generated PagL mutants in which an amino acid residue located at four extracellular loops was replaced with alanine. Apparent lipid A 3-O deacylation was observed in S. enterica expressing the recombinant mutants PagL(R43A), PagL(R44A), PagL(C85A), and PagL(R135A), but not in S. enterica expressing wild-type PagL, suggesting that the point mutations released PagL from the latency. In addition, mutations at Arg-43, Arg-44, Cys-85, and Arg-135 did not affect lipid A 3-O-deacylase activity in an S. enterica pmrA mutant or in Escherichia coli BL21(DE3). These results, taken together, indicate that specific amino acid residues located at extracellular loops of PagL are involved in the recognition of aminoarabinose-modified LPS. Furthermore, S. enterica expressing the recombinant PagL(R43A) or PagL(R135A) mutant showed apparent growth arrest at 43°C compared with S. enterica expressing wild-type PagL, indicating that the latency of PagL is important for bacterial growth.

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