Crystals of bovine trypsinogen inhibited with diisopropylfluorophosphate (DFP) were grown at pH 7 and x-ray intensity data to 2.1 Å resolution were collected. The reflections were assigned the phases determined by Kossiakoff (1977) for the isomorphous native trypsinogen structure. Using difference fourier techniques, the resulting protein structure was refined to a residual of 18.2%. As expected, a comparison between (DIP)-trypsinogen and the native trypsinogen structures shows all the structural differences to be in the catalytic site region. However, the structure of the catalytic region was expected to be quite similar to that of DFP-inhibited bovine trypsin at neutral pH, but there exist some marked differences.
In trypsin, one of the isopropyl groups of the inhibitor is analogous to the leaving group of a specific substrate and is hydrolyzed off the inhibitor. In the resulting monoisopropylphosphoryl (MIP)-trypsin, the inhibitor is oriented with a non-esterified phosphoryl oxygen in the oxyanion stabilization site as expected for a tetrahedral intermediate in proteolysis. His 57 Nε2 points toward the phosphoryl oxygen at the leaving group site, and is in a position suitable for proton donation to the leaving group.
By contrast, the inhibitor on DIP-trypsinogen remains intact and the presence of electron density at each of the three possible isopropyl locations indicates that the groups of the inhibitor are less specifically oriented. After independent refinement of each of the three possible orientations, the favored orientation was with isopropyl groups near the oxyanion and leaving group sites and the non-esterified phopsphoryl oxygen hydrogen-bonded through two solvent molecules to Ser 214 O. The imidazole ring of His 57 is displaced from its native position at the catalytic site out into the solvent region where it forms hydrogen-bonds to Tyr 94 0 and through a solvent molecule to Ser 214 O. Ser 195 Oγ has moved closer to the position occupied by the His 57 imidazole in native trypsinogen and MIP-trypsin.
Both statistical variation in orientation of the inhibitor and movement of Ser 195 Oγ toward the native imidazole site can be explained by the nonfunctionality of the oxyanion binding site in trypsinogen.
The difference between DIP-Tgen and MIP-trypsin thus suggests both non-productive transition state or substrate binding and stereochemical incompatability between the tetrahedral intermediate and the native imidazole site as possible contributors to the relative inactivity of the proenzyme.
Conversely, and of more general importance, these results emphasize the importance of exact stereochemical alignment in generating a functional catalytic enzyme.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Stroud, Robert M.}, } @phdthesis{10.7907/9psf-0a04, author = {Agard, David Andrew}, title = {Approaches to Macromolecular and Supramolecular Structure Determination}, school = {California Institute of Technology}, year = {1981}, doi = {10.7907/9psf-0a04}, url = {https://resolver.caltech.edu/CaltechTHESIS:03022018-140501638}, abstract = {All of the common techniques for obtaining structural information from macromolecular and supramolecular objects suffer from one type of drawback or another. X-ray and neutron diffraction methods have the phase problem (only the amplitude and not the phase angle of the complex diffraction vector can be experimentally observed). Low-dose 3-dimensional electron microscopy suffers from the inability to collect a complete data set due to instrumental limitations. Although light microscopy allows the sample to be kept under biological conditions (compare with standard electron microscopic techniques), the low resolution has limited its appeal to more qualitative aspects of ultrastructure analysis.
All of these limitations can be considered as a lack of information in the Fourier space domain. A generalized mathematical approach is presented where global real-space constraints are utilized to ameliorate this loss of information. In simple cases (one-dimensional analysis) it is possible to solve the phase problem, and in more complex cases (two- and three-dimensional analyses) the added real-space information is used to augment the experimentally derived data. The iterative Fourier refinement scheme was chosen as it represents the most versatile means for incorporating global knowledge concerning the real-space behavior of the object.
This approach was used to solve the three-dimensional x-ray structure of the snake polypeptide neurotoxin α-Bungarotoxin, the one dimensional transmembrane electron density profile. for acetylcholine receptor containing membranes, and to solve the missing cone problem of electron microscopic structure analysis.
Real-space constraints were also incorporated into an iterative deconvolution scheme used both for image processing and for the quantitative analysis of overlapping gel electrophoretogram peaks. None of this work would have been possible if the information supplied by real-space constraints had not been utilized . The approaches presented to these problems are general ones and should be applicable to other systems.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Stroud, Robert M.}, } @mastersthesis{10.7907/3nwd-0q45, author = {Ruark, John Edward}, title = {Structural studies of bacteriophage T7 and T7 capsids}, school = {California Institute of Technology}, year = {1978}, doi = {10.7907/3nwd-0q45}, url = {https://resolver.caltech.edu/CaltechTHESIS:03302010-083049438}, abstract = {The structures of bacteriophage T7 and T7 capsids have been investigated by small-angle X-ray scattering. Phage T7 has a radius of 301 ± 2 Å (excluding the phage tail), and a calculated volume of 1.14 ±.05 x 10^(-16) ml. The radius determined for. T7 phage in solution is about 30% greater than the radius measured from electron micrographs, indicating that considerable shrinkage and distortion occurs during sample preparation for electron microscopy. Thus the volume calculated from X-ray scattering in solution is almost twice that estimated from electron microscope measurements. Capsids that have a phage-like envelope and do not contain DNA were obtained from lysates of T7-infected E. coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA using temperature shock (capsid IV). These capsids have outer radii of 2[8?]3-289 ± 2 Å and are thus significantly smaller than the envelope of T7 phage. The thickness of the envelopes of two forms of capsid II are 20 and 23 ± 2 Å. The volume in T7 phage available to package DNA is estimated to be 9.2 ± .4 x 10^(-17) ml or 2.1 times the volume of the B form of T7 DNA. A T7 precursor capsid (capsid I), believed to be capable of pulling in DNA, has a smaller outer diameter (261 ± 2 Å) and a thicker envelope (52 ± 4 Å) than capsids II and IV. Capsid I expands when pelleted in the ultracentrifuge, but expansion can be prevented by fixing with glutaraldehyde. A low resolution model for the capsid I envelope has been developed.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Stroud, Robert M.}, } @phdthesis{10.7907/QP6S-7B65, author = {Koeppe, Roger E.}, title = {Catalysis by trypsin}, school = {California Institute of Technology}, year = {1976}, doi = {10.7907/QP6S-7B65}, url = {https://resolver.caltech.edu/CaltechTHESIS:04162010-082819644}, abstract = {Investigations are reported concerning the mechanism of catalysis by an enzyme of known structure, bovine trypsin. Evidence for proton transfers at the active site residues His 57 and Asp 102 during catalysis is provided by experimental techniques which specifically detect the ionization state of either the carboxyl group or the imidazole ring. The pK_a of Asp 102 in a chemically modified—but still active–form of trypsin is shown to be 6.8 by difference infrared spectroscopic titration. This assignment is facilitated by the use of inhibitory copper (Cu^(++)) ion which lowers the pK_(app) of Asp 102 by binding to trypsin between Asp 102 and His 57, as is demonstrated crystallographically. Information concerning the solvent accessibilities and mobilities of the three imidazole side chains of bovine trypsin is provided by measurements of the pH dependences of the rates of exchange of the ring C-2 protons with tritium in labeled water. Kinetic studies of substrate hydrolysis do not detect the pK_a of His 57 anywhere in the range 3 - 8.5. Pre-incubation of trypsin at low or at high pH shows that the failure to detect the pK_a of His 57 kinetically is not due to a slow pH-dependent conformational change. The implications of the assignment of a pK_a of 6. 8 to Asp 102, and of the pK_a of His 57 being below 3, are discussed with regard to the catalytic mechanism of the serine proteases.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Stroud, Robert M.}, } @mastersthesis{10.7907/GVT9-N257, author = {Kent, Diane Jacalyn}, title = {Electron microscope studies of chromatin subunit particles}, school = {California Institute of Technology}, year = {1975}, doi = {10.7907/GVT9-N257}, url = {https://resolver.caltech.edu/CaltechTHESIS:04202010-093957061}, abstract = {Nuclease resistant chromatin subunits (ν bodies) were isolated from rat liver chromatin after digestion with DNAase II. Electron microscope studies showed them to be similar in size to particles which have been isolated from calf thymus chromatin and also to those seen in native chromatin. ν bodies prepared from native chromatin (ν(+F1) and histone F1-depleted chromatin (ν(-F1)), as well as formaldehyde-fixed products from both these digestions (ν(FF + F1) and ν(FF-F1) were similar in size. For ν(+F1) the diameter was 80 ± 17 Å (n = 125); for ν(-F1), 87 ± 19 Å (n = 100); for ν(FF + F1), 96 ± 23 Å (n = 50); and for ν(FF – F1) 81 ± 13 Å (n = 85). Multimer ν-body fractions did not show any obvious dimers, trimers, etc. , of monomer ν bodies. Sheared, isolated chromatin from rat liver was also examined using electron microscopy and was observed as networks of long thin strands (about 15 Å wide), interspersed with regions of either coiled single strands or strands covered with protein.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Stroud, Robert M.}, }