Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
➤ Gửi thông báo lỗi ⚠️ Báo cáo tài liệu vi phạmNội dung chi tiết: Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. thermostability.Bryơn E Shaw Haribabu Arthanari \ Andrew Leee, Armando Durazo ■, Dominique p. Fruehb, Michael p. Pollastri*, Basar Bilgicera, Steven p. Gỵgid, Gerhard Wagner -, and George M. Whitesides °*’Department of Chemistry and Chemical Biology, Harvard University, Cambridge. MA., 02138:b Depa Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.rtment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School. Boston, MA.. 02115;' Department of Chemistry'- and Biochemistry;UniNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
versity of California, Los Angeles, Los Angeles. CA., 90024;d Department of Cell Biology, Harvard Medical School, Boston, MA., 02115; 'Department of CNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.dressed: bfshaw@gmwgroup.harvard.edu and gwhitesides@gmwgroup.harvard.eduAbstractThis paper combines two techniques—mass spectrometry and protein charge ladders—to examine the relationship between the surface charge and hydrophobicity of a protein (bovine carbonic anhydrase 11; BCAII) and its rate o Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.f amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-e-NH3* groups—a typNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
e of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its 2° or 3° structure—resultedNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.ve acetylation produced BCAII proteins with one additional hydrogen protected after two hours in deuterated buffer (pD 7.4, 15 °C). NMR spectroscopy demonstrated that these protected hydrogen atoms were not located on the side chain of the acetylated lysine residues (i.e., lys-e-NHCOCHẠ The decrease Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs were the least thermostablNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
e (as measured by differential scanning calorimetry). The fact that the rates of HD exchange were similar for perbutyrated BCAII (e.g., (lys-e-NHC0(CHNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.etermining the rate of H/D exchange. These kinetic electrostatic effects could complicate the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the p Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.rotein to an oligonucleotide or another charged ligand or protein).Key words: amide H/D exchange, lysine acetylation, mass spectrometry, protein foldiNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
ng, carbonic anhydrase II, protein charge ladder, hydrogen/deuterium, electrostatic potential.2IntroductionWe wished to determine how the surface charNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.n-dcutcrium (ll'D) exchange of die rungs (successively acylated sets of proteins) of two protein charge ladders1 J with electro-spray ionization mass spectrometry (LS1-MS). A “protein charge ladder” is a mixture of charge isomers generated by the modification of the functional groups of a protein, r Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.he charge ladders we used were prepared by sequentially acylating all 18 lysine-c-Nllj’ of bovine carbonic anhydrase If’ (BCA IT) with acetic or butyrNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
ic anhydride to yield lysine-e-NHCOCHj and lysine-e-NHC.C)(CH )2CH„The isoelec trie point (pl) of BC.A ĨĨ is ~ 5.9. Previous experiments al pH 8.4 havNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.lue of -1.0 that might be expected for -NH;T -» -NHCOCHj can be explained by charge regulation ' (e.g., the electrostatic effect of acylating -E-NH3* is not limited to the E-nitrogen that is modified). Solvent ions, for example, will reorganize around the E-nitrogen, and the values of pKi of nearby Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.ionizable groups will adjust to the new electrostatic environment that results from neutralization of the lysine C-NH/ group. The BCA11 charge ladderNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
contains 19 charge isomers or "Rings,” and therefore spans approximately 16 units of charge. The acetylation of all 18 lysine residues (peracetylationNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.trometry established a linear relationship between the net negative charge of folded BCA TĨ (e.g., the number of acylations) and the number ol hydrogens that do not exchange with solvent al ter a 2 h incubation in deuterated buffer (we say these hydrogen are protected from exchange). The acetylation Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. of each lysine, lor example, generated approximately one additional hydrogen that was protec led from H'T) exchange after 2 h (at 15 °C. pD 7.4). MulNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
ti-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy demonstrated that the additional protected hydrogen atoms were nor located on the lysine-Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.e ladder3had the slowest rates of global5 HD exchange, an analysis with differential scanning calorimetry showed that these rungs also had lower conformational stability than the lower rungs.Hydrogen Exchange as a Tool for Studying the Structure and Folding of Proteins. The rate at which a protein e Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.xchanges its backbone amide hydrogens with tritium or deuterium in buffer has been used for nearly 60 years10'11 to study the structure 12'13, foldingNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
14» and conformational stability15"1- of proteins. In fact, the first measurements of H. D exchange were not made with any form of spectroscopy, but Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.d then dried under vacuum with P2O5).11 The utility of hydrogen exchange in protein biochemistry is based upon the generally observed correlation between the rate of amide hydrogen exchange and i) the rate of protein folding, ii) the local structure surrounding a backbone amide, and iii) the conform Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.ational stability of the folded protein.1'18 In spite of the historic and now widespread use of hydrogen exchange in structural biology and biochemistNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
ry—and in spite of all that is known about the processes of HD exchange in proteins—the reasons for why many amide hydrogen atoms are slow to exchangeNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. below).The exchange of amide hydrogens with aqueous solvent is catalyzed by both acid and base, and the minimum rate of exchange for an unstructured polypeptide occurs at ~ pH 2.5?3 Above pH 4, the primary catalyst for amide hydrogen exchange is hydroxide 24 (the pKa of the backbone amide in an uns Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.tructured polypeptide is ~ 15); below pH 4, the exchange is catalyzed by hydronium. In the case of an unstructured polypeptide, the exchange of amideNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
hydrogen with solvent is fast: it occurs in milliseconds to seconds at pH 7 and room temperature.25 With a folded or structured protein, however, the Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.sed for decades to understand the kinetics of amide hydrogen exchange in folded proteins.11'284This model (summarized in Equation 1) involves a transition between two states: "open" and “closed". Hydrogen exchange occurs in the “open” Slate and not in the "closed” state.* *«NH . .5=*: NH —; K ~ kjk. Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. (1) ckncu -openopen op op cl ' '"dIn Equation 1, klnl refers to the rate constant for the exchange of an amide in an unstructured polypeptide (i.e.,Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
the intrinsic rate of exchange): kcl refers to the rate constant for a closing reaction (e.g.. refolding or a change in conformation). The intrinsic rNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.cheme in (1) can occur at two extremes: i) kci » k-,; that is, the closing reaction (such as folding or a change in conformation) is much faster than the intrinsic rate of exchange; and: ii) ke.<Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability.
opThe two most widely accepted theories for understanding how various amides undergo hydrogen exchange in proteins are known as “local unfolding” 18 2Gọi ngay
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