scholarly journals In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8

ACS Omega ◽  
2017 ◽  
Vol 2 (8) ◽  
pp. 4991-4999 ◽  
Author(s):  
Patricia Wennerstrand ◽  
Annica Blissing ◽  
Lars-Göran Mårtensson
Keyword(s):  
Protein Stability ◽  
Biophysical Characterization ◽  
Naturally Occurring ◽  
Vitro Protein

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5185 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Sara Züger ◽  
Edith Buchinger ◽  
Hideo Iwaï
Keyword(s):  
Protein Ligation ◽  
Naturally Occurring ◽  
Vitro Protein

Differential Regulation of Articular Cartilage Biomechanical and Biochemical Properties by IGF-1 and TGF-β1 During In Vitro Growth

2009 ◽  
Author(s):  
Michael E. Stender ◽  
Christian R. Flores ◽  
Kristin J. Dills ◽  
Gregory M. Williams ◽  
Kevin M. Stewart ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Growth Models ◽  
Biochemical Properties ◽  
Low Friction ◽  
Cartilage Growth ◽  
Detailed Characterization ◽  
Naturally Occurring ◽  
Tgf Β1

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is ineffective. Thus, there is a desire to engineer effective replacement tissue that could be used for AC repair. Previous studies [1] have shown that culture of immature cartilage with medium including TGF-β1 will result in a more mature tissue than culture with IGF-1. Detailed characterization of tissue mechanical properties would be helpful for development of cartilage growth models [2].

scholarly journals Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5α Protein

2008 ◽  
Vol 82 (23) ◽  
pp. 11669-11681 ◽  
Author(s):  
Alak Kanti Kar ◽  
Felipe Diaz-Griffero ◽  
Yuan Li ◽  
Xing Li ◽  
Joseph Sodroski
Keyword(s):  
Variable Region ◽  
Globular Structure ◽  
Biophysical Characterization ◽  
Immunodeficiency Virus ◽  
Infected Insect ◽  
Low Levels ◽  
Trim Proteins

ABSTRACT The tripartite motif (TRIM) protein, TRIM5α, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5α promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5α protein (TRIM5Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.

scholarly journals Biophysical characterization of naturally occurring titin M10 mutations

2015 ◽  
Vol 24 (6) ◽  
pp. 946-955 ◽  
Author(s):  
Michael W. Rudloff ◽  
Alec N. Woosley ◽  
Nathan T. Wright
Keyword(s):  
Biophysical Characterization ◽  
Naturally Occurring

scholarly journals Isolation, Synthesis, and Antimicrobial Activities of Naturally Occurring θ-Defensin Isoforms from Baboon Leukocytes

2008 ◽  
Vol 76 (12) ◽  
pp. 5883-5891 ◽  
Author(s):  
Angie E. Garcia ◽  
George Ösapay ◽  
Patti A. Tran ◽  
Jun Yuan ◽  
Michael E. Selsted
Keyword(s):  
Bone Marrow ◽  
Single Species ◽  
Antimicrobial Activities ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Naturally Occurring ◽  
Test Organisms ◽  
High Degree

ABSTRACT θ-Defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon θ-defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding θ-defensin precursors were characterized, allowing for the prediction of 10 theoretical θ-defensins (BTD-1 to BTD-10) produced by binary, head-to-tail splicing of nonapeptides excised from paired precursors. Five of the predicted θ-defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti-θ-defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the θ-defensins tested. Thus, baboons express numerous θ-defensins which appear to differentially contribute to host defense against diverse pathogens.

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