Sugar transport by the bacterial phosphotransferase system. Radioactive and electron paramagnetic resonance labeling of the Salmonella typhimurium phosphocarrier protein (HPr) at the NH2-terminal methionine.

H Grill,N Weigel,B J Gaffney,S Roseman

doi:10.1016/s0021-9258(19)45408-2

Abstract

Sugar transport by the bacterial phosphotransferase system. Radioactive and electron paramagnetic resonance labeling of the Salmonella typhimurium phosphocarrier protein (HPr) at the NH2-terminal methionine.

Highlights

Investigation of the complex interactions between proteinHPr' from Salmonella typhimurium
Since S. typhimurium HPr contains only 2 methionine resi- Synthesis of Radioactive HPr-['HIMethyl iodide was disdues,onelocated at the NH, terminus,andtheotherat tilled to the bottom of a break-seal ampule by warming the residue 80 (84 total residues), it seemed possible that this upper portion while the bottom was maintained in a mixture procedure would give limited derivatization with retentionof of dry ice and methylCellosolve
For reasons given inthe introductory section,the availability of HPr derivatized with different probes will greatly facilitate studies aimed at understanding how individual proteins of the phosphotransferase ase system (PTS) interact with one another, with membrane components, and whether, for example, theyattachanddetach from the membrane components during sugar transport in vesicles

Summary

Introduction

Investigation of the complex interactions between proteinHPr' from Salmonella typhimurium. Low molecular weight phosphocarrier HPr. Salmonella Current studies in this laboratory are concerned typhimurium HPr contains two methionine residues, with characterizing the individual components of the PTS, one at the NH2terminus, and the other at amino acid and with their interactions in vitro and in natural memTwraw2Hae,cnhiPas5tdteiis,hrdv5SEwuuet-htsPetaiaeneeRsrd8tkpEp-0rlt,hu.PaaooGTmrbRiesae.hftpiplleRehekhrydd.myoolb(lrtH-eayo1elttP9l,h-ea6rpa3ot8Hy-ipdwo)r[pPnJr(oaao2rf.sarl-wBLesiibsnsidirioonatitohhllkynm.aoyso[tmaClae3inoncdhHoxdeevgy]itmnmSileatT.nmrtbeaioh2eterd4iehatkto3yytsr),el(,amnirLadoeaniitdttnodh1ihivkldy0aa,el8enwbT]2,He-a9-.al2Ps15Pnea,0%rdd.-s,.88)uptpmobhfsrhroaeeaottynnhteHrneeiiannePnvsPrtres,eTtafsrhseinaSucarcdlctetwroheswwti.aalhlciesFettrhoirHecoprqaonPrtunnsohr.ietereeaeUsaxinennalsdtmtoesiumxtEidhnpayeneetrzesroypiplfmybermo,ectehefipaInfoeei,trccrsoeiaot,nrilmeailtytesnHprdilPaosleafcdrtbateutdieahorlvunieennnaddspgnIdiInrtoeItoafGrhgtseep1tesai"aop(unniouhcsrdrssohinsaItoo-aIgsfpurity, contained no native HPr, and showed 80 -+ 7% AMs"),groups of three proteins(e.g.Enzyme I, HPr, and1IIG1', of the activity of the nativeprotein. Since the EPR probe is close to theactive site histidine (residue 15), it seems possible that the EPR-labeled HPr will give increased rotational correlation times in the presence of other PTS proteins, such as Enzyme I and IIIG'",despite the segmental flexibility, and preliminary experiments indicate this to be the case. For this type of experiment, radiolabeled HPr would provide thenecessary sensitivity and accuracy. This article must be hereby marked "aduertisement" in accord-

Methods

Results

Conclusion