Thomas K. Harris

High‐Precision Measurement of Hydrogen Bond Lengths in Proteins by Nuclear Magnetic Resonance Methods

We have compared hydrogen bond lengths on enzymes derived with high precision (≤ ±0.05 Å) from both the proton chemical shifts (δ) and the fractionation factors (ϕ) of the proton involved with those obtained from protein X‐ray crystallography. Hydrogen bond distances derived from proton chemical shifts were obtained from a correlation of 59 O—H····O hydrogen bond lengths, measured by small molecule high‐resolution X‐ray crystallography, with chemical shifts determined by solid‐state nuclear magnetic resonance (NMR) in the same crystals (McDermott A, Ridenour CF, Encyclopedia of NMR, Sussex, U.K.: Wiley, 1996:3820–3825). Hydrogen bond distances were independently obtained from fractionation factors that yield distances between the two proton wells in quartic double minimum potential functions (Kreevoy MM, Liang TM, J Am Chem Soc, 1980;102:3315–3322). The high‐precision hydrogen bond distances derived from their corresponding NMR‐measured proton chemical shifts and fractionation factors agree well with each other and with those reported in protein X‐ray structures within the larger errors (±0.2–0.8 Å) in distances obtained by protein X‐ray crystallography. The increased precision in measurements of hydrogen bond lengths by NMR has provided insight into the contributions of short, strong hydrogen bonds to catalysis for several enzymatic reactions. Proteins 1999;35:275–282.

Short, strong hydrogen bonds on enzymes: NMR and mechanistic studies

The lengths of short, strong hydrogen bonds (SSHBs) on enzymes have been determined with high precision (^0.05 A ˚ ) from the chemical shifts (d ), and independently from the D/H fractionation factors (f ) of the highly deshielded protons involved. These H-bond lengths agree well with each other and with those found by protein X-ray crystallography, within the larger errors of the latter method (^ 0.2 to ^ 0.8 A ˚ ) (Proteins 35 (1999) 275). A model dihydroxynaphthalene compound shows a SSHB of 2:54 ^ 0:04 Abased on d ¼ 17:7 ppm and f ¼ 0:56 ^ 0:04; in agreement with the high resolution X-ray distance of 2:55 ^ 0:06 A ˚ . On ketosteroid isomerase, a SSHB is found ð2:50 ^ 0:02 AÞ; based on d ¼ 18:2 ppm and f ¼ 0:34; from Tyr- 14 to the 3-O 2 of estradiol, an analog of the enolate intermediate. Its strength is , 7 kcal/mol. On triosephosphate isomerase, SSHBs are found from Glu-165 to the 1-NOH of phosphoglycolohydroxamic acid (PGH), an analog of the enolic intermediate ð2:55 ^ 0:05 AÞ; and from His-95 to the enolic-O 2 of PGH ð2:62 ^ 0:02 AÞ: In the methylglyoxal synthase - PGH complex, a SSHB ð2:51 ^ 0:02 AÞ forms between Asp-71 and the NOH of PGH with a strength of

Thermodynamically balanced inside‐out (TBIO) PCR‐based gene synthesis: a novel method of primer design for high‐fidelity assembly of longer gene sequences

4.7 kcal/mol. When serine proteases bind mechanism-based inhibitors which form tetrahedral Ser-adducts analogous to the tetrahedral intermediates in catalysis, the Asp· · ·His H-bond of the catalytic triad becomes a SSHB (Proc. Natl Acad. Sci. USA 95 (1998) 14664), 2.49- 2.63 Ain length. Similarly, on the serine-esterase, butyrylcholinesterase complexed with the mechanism-based inhibitor m-(N,N,N- trimethylammonio)-2,2,2-trifluoroacetophenone, a SSHB forms between Glu-327 and His-438 of the catalytic triad, 2:61 ^ 0:04 Ain length, based on d ¼ 18:1 ppm and f ¼ 0:65 ^ 0:10: Very similar results are obtained with (human) acetylcholinesterase. The strength of this SSHB is at least 4.9 kcal/mol. q 2002 Elsevier Science B.V. All rights reserved.