scholarly journals Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm

2021 ◽  
Author(s):  
Masaki Sasai
Keyword(s):  
Temperature Compensation ◽  
Oscillation Amplitude ◽  
Thermal Fluctuations ◽  
Reaction Rates ◽  
System Level ◽  
Single Amino Acid ◽  
Structural Transitions ◽  
Single Amino Acid Residue ◽  
Residue Substitution

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. We analyzed this temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model suggests that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

scholarly journals A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function

2009 ◽  
Vol 106 (17) ◽  
pp. 6939-6944 ◽  
Author(s):  
Jingyan Fu ◽  
Minglei Bian ◽  
Junjun Liu ◽  
Qing Jiang ◽  
Chuanmao Zhang
Keyword(s):  
Amino Acid ◽  
Aurora Kinase ◽  
Single Amino Acid ◽  
Aurora B ◽  
Aurora A ◽  
Single Amino Acid Residue ◽  
Single Amino Acid Change ◽  
Equivalent Residue ◽  
And Function

Aurora kinase-A and -B are key regulators of the cell cycle and tumorigenesis. It has remained a mystery why these 2 Aurora kinases, although highly similar in protein sequence and structure, are distinct in subcellular localization and function. Here, we report the striking finding that a single amino acid residue is responsible for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa cells, Aurora-AG198N was recruited to the inner centromere in metaphase and the midzone in anaphase, reminiscent of the Aurora-B localization. Moreover, Aurora-AG198N compensated for the loss of Aurora-B in chromosome misalignment and cell premature exit from mitosis. Furthermore, Aurora-AG198N formed a complex with the Aurora-B partners, INCENP and Survivin, and its localization depended on this interaction. Aurora-AG198N phosphorylated the Aurora-B substrates INCENP and Survivin in vitro. Therefore, we propose that the presence of Gly or Asn at a single site assigns Aurora-A and -B to their respective partners and thus to their distinctive subcellular localizations and functions.

scholarly journals The Capsid Proteins of Aleutian Mink Disease Virus Activate Caspases and Are Specifically Cleaved during Infection

2009 ◽  
Vol 84 (6) ◽  
pp. 2687-2696 ◽  
Author(s):  
Fang Cheng ◽  
Aaron Yun Chen ◽  
Sonja M. Best ◽  
Marshall E. Bloom ◽  
David Pintel ◽  
...  
Keyword(s):  
Virus Infection ◽  
Disease Virus ◽  
Virus Production ◽  
Capsid Proteins ◽  
Single Amino Acid ◽  
Aleutian Mink Disease Virus ◽  
Single Amino Acid Residue ◽  
Caspase 7

ABSTRACT Aleutian mink disease virus (AMDV) is currently the only known member of the genus Amdovirus in the family Parvoviridae. It is the etiological agent of Aleutian disease of mink. We have previously shown that a small protein with a molecular mass of approximately 26 kDa was present during AMDV infection and following transfection of capsid expression constructs (J. Qiu, F. Cheng, L. R. Burger, and D. Pintel, J. Virol. 80:654-662, 2006). In this study, we report that the capsid proteins were specifically cleaved at aspartic acid residue 420 (D420) during virus infection, resulting in the previously observed cleavage product. Mutation of a single amino acid residue at D420 abolished the specific cleavage. Expression of the capsid proteins alone in Crandell feline kidney (CrFK) cells reproduced the cleavage of the capsid proteins in virus infection. More importantly, capsid protein expression alone induced active caspases, of which caspase-10 was the most active. Active caspases, in turn, cleaved capsid proteins in vivo. Our results also showed that active caspase-7 specifically cleaved capsid proteins at D420 in vitro. These results suggest that viral capsid proteins alone induce caspase activation, resulting in cleavage of capsid proteins. We also provide evidence that AMDV mutants resistant to caspase-mediated capsid cleavage increased virus production approximately 3- to 5-fold in CrFK cells compared to that produced from the parent virus AMDV-G at 37°C but not at 31.8°C. Collectively, our results indicate that caspase activity plays multiple roles in AMDV infection and that cleavage of the capsid proteins might have a role in regulating persistent infection of AMDV.

scholarly journals Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution

2005 ◽  
Vol 280 (25) ◽  
pp. 24104-24112 ◽  
Author(s):  
Shigetarou Mori ◽  
Shigeyuki Kawai ◽  
Feng Shi ◽  
Bunzo Mikami ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Single Amino Acid ◽  
Nad Kinase ◽  
Single Amino Acid Residue ◽  
Residue Substitution ◽  
Nadh Kinase

scholarly journals Single residue substitution in protamine 1 disrupts sperm genome packaging and embryonic development in mice

2021 ◽  
Author(s):  
Lindsay Moritz ◽  
Samantha Schon ◽  
Mashiat Rabbani ◽  
Yi Sheng ◽  
Devon Pendlebury ◽  
...  
Keyword(s):  
Protein Function ◽  
Single Amino Acid ◽  
Genome Packaging ◽  
Post Translational Modifications ◽  
Dna Compaction ◽  
Arginine Residues ◽  
Residue Substitution ◽  
Protamine 1

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, an alanine substitution (P1 K49A) results in ectopic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin. In vitro, the P1 K49A substitution decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential to ensure reproductive fitness.

scholarly journals Biochemical Analysis of Phytolacca DOPA Dioxygenase

2015 ◽  
Vol 10 (5) ◽  
pp. 1934578X1501000
Author(s):  
Kana Takahashi ◽  
Kazuko Yoshida ◽  
Kei Yura ◽  
Hiroshi Ashihara ◽  
Masaaki Sakuta
Keyword(s):  
Biochemical Analysis ◽  
Structural Model ◽  
Specific Activity ◽  
Three Dimensional ◽  
Single Amino Acid ◽  
Phytolacca Americana ◽  
Conserved Motif ◽  
Single Amino Acid Residue ◽  
The Difference

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD1. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity

2010 ◽  
Vol 391 (6) ◽  
Author(s):  
Sascha N. Stumpp ◽  
Bianca Heyn ◽  
Susanne Brakmann
Keyword(s):  
Viral Replication ◽  
Reverse Transcriptase ◽  
Error Rate ◽  
Antiviral Treatment ◽  
Rnase H ◽  
Single Amino Acid ◽  
Selection Scheme ◽  
Single Amino Acid Residue ◽  
Hiv 1

AbstractHIV-1 reverse transcriptase (HIV-1 RT) copies the RNA genome of HIV-1 into DNA, thereby committing errors at an exceptionally high frequency. Viral offspring evolve rapidly and consequently are capable of evading the immune response as well as antiviral treatment. However, error-prone viral replication could drive HIV close to extinction owing to an intolerable load of deleterious mutations. We applied a genetic selection scheme to identify variants of HIV-1 RT with a further increased error rate to study the relationship between error rate and viral replication. Using this approach, we identified 16 mutator candidates, two of which were purified and further studiedin vitro. One of these variant enzymes showed a generally increased mutation frequency as compared with the reference enzyme. A single amino acid residue, R448, is probably responsible for the observed effect. Mutation of this residue, which is located within the RNase H domain of HIV-1 RT, seems to perturb the interaction with template RNA and consequently affects polymerase activity and fidelity.

scholarly journals Tuning the circadian period of cyanobacteria up to 6.6 days by the single amino acid substitutions in KaiC

2020 ◽  
Vol 117 (34) ◽  
pp. 20926-20931
Author(s):  
Kumiko Ito-Miwa ◽  
Yoshihiko Furuike ◽  
Shuji Akiyama ◽  
Takao Kondo
Keyword(s):  
Amino Acid ◽  
Atpase Activity ◽  
Temperature Compensation ◽  
Period Change ◽  
Single Amino Acid ◽  
Circadian Period ◽  
Clock Proteins ◽  
Wide Range

The circadian clock of cyanobacteria consists of only three clock proteins, KaiA, KaiB, and KaiC, which generate a circadian rhythm of KaiC phosphorylation in vitro. The adenosine triphosphatase (ATPase) activity of KaiC is the source of the 24-h period and temperature compensation. Although numerous circadian mutants of KaiC have been identified, the tuning mechanism of the 24-h period remains unclear. Here, we show that the circadian period of in vitro phosphorylation rhythm of mutants at position 402 of KaiC changed dramatically, from 15 h (0.6 d) to 158 h (6.6 d). The ATPase activities of mutants at position 402 of KaiC, without KaiA and KaiB, correlated with the frequencies (1/period), indicating that KaiC structure was the source of extra period change. Despite the wide-range tunability, temperature compensation of both the circadian period and the KaiC ATPase activity of mutants at position 402 of KaiC were nearly intact. We also found that in vivo and in vitro circadian periods and the KaiC ATPase activity of mutants at position 402 of KaiC showed a correlation with the side-chain volume of the amino acid at position 402 of KaiC. Our results indicate that residue 402 is a key position of determining the circadian period of cyanobacteria, and it is possible to dramatically alter the period of KaiC while maintaining temperature compensation.

scholarly journals Characterization of LytA-Like N-Acetylmuramoyl-l-Alanine Amidases from Two New Streptococcus mitis Bacteriophages Provides Insights into the Properties of the Major Pneumococcal Autolysin

2004 ◽  
Vol 186 (24) ◽  
pp. 8229-8239 ◽  
Author(s):  
Patricia Romero ◽  
Rubens López ◽  
Ernesto García
Keyword(s):  
Amino Acid ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Lytic Phage ◽  
Streptococcus Mitis ◽  
Single Amino Acid Residue ◽  
Low Activity

ABSTRACT Two new temperate bacteriophages exhibiting a Myoviridae (φB6) and a Siphoviridae (φHER) morphology have been isolated from Streptococcus mitis strains B6 and HER 1055, respectively, and partially characterized. The lytic phage genes were overexpressed in Escherichia coli, and their encoded proteins were purified. The lytA HER and lytA B6 genes are very similar (87% identity) and appeared to belong to the group of the so-called typical LytA amidases (atypical LytA displays a characteristic two-amino-acid deletion signature). although they exhibited several differential biochemical properties with respect to the pneumococcal LytA, e.g., they were inhibited in vitro by sodium deoxycholate and showed a more acidic pH for optimal activity. However, and in sharp contrast with the pneumococcal LytA, a short dialysis of LytAHER or LytAB6 resulted in reversible deconversion to the low-activity state (E-form) of the fully active phage amidases (C-form). Comparison of the amino acid sequences of LytAHER and LytAB6 with that of the pneumococcal amidase suggested that Val317 might be responsible for at least some of the peculiar properties of S. mitis phage enzymes. Site-directed mutagenesis that changed Val317 in the pneumococcal LytA amidase to a Thr residue (characteristic of LytAB6 and LytAHER) produced a fully active pneumococcal enzyme that differs from the parental one only in that the mutant amidase can reversibly recover the low-activity E-form upon dialysis. This is the first report showing that a single amino acid residue is involved in the conversion process of the major S. pneumoniae autolysin. Our results also showed that some lysogenic S. mitis strains possess a lytA-like gene, something that was previously thought to be exclusive to Streptococcus pneumoniae. Moreover, the newly discovered phage lysins constitute a missing link between the typical and atypical pneumococcal amidases known previously.

scholarly journals Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)

2007 ◽  
Author(s):  
◽  
Sarveshni Pillay
Keyword(s):  
Recombinant Dna ◽  
Paper Industry ◽  
Residual Activity ◽  
Industrial Applications ◽  
Thermomyces Lanuginosus ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Residue ◽  
Recombinant Dna Techniques

Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

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