For Table <strong>of</strong> Contents Use OnlypressureManuscript Title: <strong>Structural</strong> <strong>and</strong> <strong>Thermodynamic</strong> <strong>Characterization</strong> <strong>of</strong> <strong>T4</strong> <strong>Lysozyme</strong> Mutants <strong>and</strong> the Contribution <strong>of</strong>Internal Cavities to Pressure DenaturationAuthors: Nozomi Ando, Buz Barstow, Walter A. Baase, Andrew Fields, Brian W. Matthews, <strong>and</strong> Sol M. Gruner50
Supporting Online MaterialSupporting Material 1: Composition <strong>of</strong> Cell Culture BrothsProteins used in this work were expressed in bacteria grown in media similar incomposition, but not identical to st<strong>and</strong>ard Luria-Bertani broth or M9a media. The mediareferred to as modified Luria-Bertani broth contains 12 g tryptone, 10 g NaCl, 1 gglucose <strong>and</strong> 5 g yeast extract per liter <strong>of</strong> broth. LBH broth contains 10 g tryptone, 5 gNaCl, 1 ml <strong>of</strong> 1 N NaOH <strong>and</strong> 5 g yeast extract per liter <strong>of</strong> broth. The version <strong>of</strong> M9amedia used for these experiments contains 7 g Na 2 HPO 4· 7H 2 O, 3 g KH 2 PO 4 , 1 g NH 4 Cl,0.5 g NaCl mixed with one liter <strong>of</strong> water <strong>and</strong> autoclaved, followed by sterile addition <strong>of</strong>10 mL 20% glucose, 0.4 mL 0.25 M CaCl 2 , 1 mL 1 M MgSO 4 , <strong>and</strong> 2 mL 0.5 mg/mLthiamine.Supporting Material 2: Production <strong>of</strong> L99A, L99G/E108V, <strong>and</strong> A98L mutantsThe L99A, L99G/E108V, <strong>and</strong> A98L mutants were expressed <strong>and</strong> purified by a modifiedversion <strong>of</strong> the protocol described by Poteete, et al. (1). For clarity, the entire procedure islisted here. E. coli strain RR1 containing lysozyme-producing plasmids were streakedfrom frozen cultures onto modified LB-agar plates containing 100 µg/mL ampicillin.Single colonies were used to inoculate 100 mL cultures <strong>of</strong> LBH broth (Supportingmaterial 1) containing 200 µg/mL ampicillin, <strong>and</strong> were incubated overnight at 32˚ C. The100 mL culture was diluted into four 1 L cultures <strong>of</strong> LBH broth in Fernbach flasks withaeration. Protein expression was induced by an IPTG concentration <strong>of</strong> 180 mg/L at anoptical density <strong>of</strong> 0.6. After 90 minutes the cultures were pelleted <strong>and</strong> resuspended in 100mL <strong>of</strong> 50 mM Tris-HCl, pH 7.5, 10 mM Na 3 HEDTA, 0.1% triton X-100 buffer with 1protease inhibitor tablet (Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets,
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