BtuF　is　the　periplasmic　binding　protein　(PBP)　that　binds　vitamin　B-12　and　delivers　it　to　the　periplasmic　surface　of　the　ABC　transporter　BtuCD.　PBPs　generally　exhibit　considerable　conformational　changes　during　ligand　binding　process,　however,　BtuF　belongs　to　a　subclass　of　PBPs　that,　doesn＇t　show　such　behavior　on　the　basis　of　the　crystal　structures.　Employing　steered　molecular　dynamics　on　the　B-12-bound　BtuF,　we　investigated　the　energetics　and　mechanism　of　BtuF.　A　potential　of　mean　force　along　the　postulated　vitamin　B-12　unbinding　pathway　was　constructed　through　Jarzynski＇s　equality.　The　large　free　energy　differences　of　the　postulated　B-12　unbinding　process　suggests　the　B-12-bound　structure　is　in　a　stable　closed　state　and　some　conformation　changes　may　be　necessary　to　the　B-12　unbinding.　From　the　result　of　the　principal　component　analysis,　we　found　the　BtuF-B-12　complex　shows　clear　opening-closing　and　twisting　motion　tendencies　which　may　facilitate　the　unbinding　of　B-12　from　the　binding　pocket.　The　intrinsic　flexibility　of　BtuF　was　also　explored,　and　it＇s　suggested　the　Trp44-Gln45　pair,　which　is　situated　at　the　mouth　of　the　B-12　binding　pocket,　may　act　as　a　gate　in　the　B12　binding　and　unbinding　process.　(c)　2008　Elsevier　B.V.　All　rights　reserved.