Warum sind Proteine geladen? Netzwerke aus Ladungs‐Ladungs‐Wechselwirkungen in Proteinen, analysiert über Ladungsleitern und Kapillarelektrophorese

Abstract

Fast alle Proteine enthalten geladene Aminosäuren. Während für einzelne Ladungen im aktiven Zentrum die Funktion bei der Katalyse oder der Bindung häufig aufgeklärt werden kann, ist es weniger ersichtlich, welche Funktion den Ladungen außerhalb dieser Region zuzuordnen ist. Sind sie für die Löslichkeit notwendig? Oder haben sie andere Aufgaben? Lassen sich durch Verändern dieser Ladungen die Proteineigenschaften variieren? Eine Kombination aus Kapillarelektrophorese (CE) und “Proteinladungsleitern” ermöglicht es, die Funktionen geladener Reste auf der Proteinoberfläche außerhalb des aktiven Zentrums zu studieren. Dazu werden diese Reste durch Reaktionen modifiziert, die eine große Zahl unterschiedlich geladener Derivate des Proteins liefern. CE trennt diese Derivate in Fraktionen mit der gleichen Zahl modifizierter geladener Gruppen. Die Untersuchung des Ladungseinflusses auf die Proteineigenschaften unter Verwendung von Ladungsleitern ermöglicht es, die Nettoladung und den hydrodynamischen Radius der Proteine zu bestimmen und auf die Rolle geladener Reste bei Ligandbindung und Proteinfaltung zu schließen.

What Are We Missing by Not Measuring the Net Charge of Proteins?

“…[18,19] In contrast, the capillary electrophoresis of a" protein charge ladder" requires nanoliters of protein solution, at micromolar concentration and < 10 minutes per experiment. [1,2,4,6] We estimate that the net charge of only about 0.1 %o ft he approximately7 0000 unique proteins currently in the Protein Data Bank have been measured in their folded state at pH ¼ 6 pI. Of course,i soelectricp oints can be measured for thousands of proteins in as ingle experiment, but isoelectric points are poor expressions of net charge.T he pI of ap rotein represents only one value of Z at one value of pH (i.e., Z = 0a tp H= pI).…”

“…This Concept article discusses how an overlooked tool in analytical chemistry,t he "protein chargel adder," [1][2][3][4] is beginning to provide new quantitative information about the fundamental electrostatic properties of metalloproteins. [5][6][7] When analyzed with capillary zone electrophoresis (CZE or CE), a"protein charge ladder" (Figure 1A,B) is one of the few tools that can rapidly quantify the net electrostatic charge(Z)ofafolded pro-tein in solution.…”

“…[13] The net charge of af olded protein in water is ap roperty that is best measured, not computationally simulated. [1] Quantifying the actual net charge of af olded protein in solution has been historically impractical. [1,14] Science has enjoyed ah alf-century boom in the development and optimization of tools to measure ap rotein mass-and mass spectrometry journals to publish the measurements-but there have been few tools developed to measure the net charge of folded, solvated proteins at pH ¼ 6 pI.…”

“…[1] Quantifying the actual net charge of af olded protein in solution has been historically impractical. [1,14] Science has enjoyed ah alf-century boom in the development and optimization of tools to measure ap rotein mass-and mass spectrometry journals to publish the measurements-but there have been few tools developed to measure the net charge of folded, solvated proteins at pH ¼ 6 pI. Some of the earliest measurements of protein net charge using pH titrationsr elied upon inaccurate assumptions that only H + and OH À ions would bind reversiblyt ot he protein (ignoring other tightly bound cationsa nd anions).…”

“…Proteins that have identical isoelectricp oints can have different values of Z at pH ¼ 6 pI. [1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering.…”