摘要:

AbstractA peptide corresponding to the three zinc finger domains of the human transcription factor Sp1 has been expressed and found to bind a consensus Sp1 binding site with the sequence 5′‐GGGGCGGGG‐3′. Examination of the amino acid distributions within a large zinc finger sequence data base and chemical arguments suggested that a particular Arg to Gln sequence change might convert binding specificity to 5′‐GGGGCAGGG‐3′. Experimental tests of this hypothesis revealed that such a change could be induced only when two other sequence changes, deduced from examination of sequence correlations, were made as well. These results provide the most direct information to date about how zinc finger proteins might recognize adenine‐containing binding sites and bear on the existence and nature of any code between zinc finger protein and bindi

ISSN:0887-3585    

DOI:10.1002/prot.340120202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWhat is the current state of the art in protein design? This question was approached in a recent two‐week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four‐stranded β‐sheets, a scaffold on which to explore variations in loop topology; Grendel, a four‐helical membrane anchor, ready for fusion to water‐soluble functional domains; Fingerclasp, a dimer of interdigitating β–β–α units, the simplest variant of the “handshake” structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather—a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three‐dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible

ISSN:0887-3585    

DOI:10.1002/prot.340120203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to α‐helical conformations. The α‐helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectro

ISSN:0887-3585    

DOI:10.1002/prot.340120204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose‐1,5‐bis‐phosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2‐type enzyme from the photosynthetic bacteriumRhodospirillum rubrumshows that the core of the dimer, consisting of the two C‐terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N‐terminal domains have mainly negative potential. At the active site situated between the C‐terminal domain of one subunit and the N‐terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pKvalue for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the ε‐amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pKof K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2building block of the L8S8‐type Rubisco from spinach is, despite only 30% amino acid homology for the L‐chains, strikingly similar to that of the L2‐type Rubisco fromRhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8‐type enzymes. The higher potential at this site might be due to two remote histidine residues, which are cons

ISSN:0887-3585    

DOI:10.1002/prot.340120205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractLead has been used as a substitute for calcium binding to tobacco mosaic virus (TMV). The high atomic number of lead has allowed us to use difference maps from X‐ray fiber diffraction data to characterize a calcium‐binding site in the virus. The metal ligands are slightly different from those previously believed to bind calcium to TMV, although the binding site is very close to one previously described. Two acetate groups are also bound to the lead atom. There is no significant backbone conformational change in the protein as a result of metal binding; the binding is accomplished by means of relatively small movements in amino acid side cha

ISSN:0887-3585    

DOI:10.1002/prot.340120206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractPig muscle phosphoglycerate kinase has been crystallized from polyethyleneglycol in the presence of its substrate 3‐phospho‐D‐glycerate (3‐PG) and the structure has been determined at 2.0 Å resolution. The structure was solved using the known structure of the substrate‐free horse muscle enzyme and has been refined to a crystallographicR‐factor of 21.5%. 3‐Phospho‐D‐glycerate is bound to the N‐domain of the enzyme through a network of hydrogen bonds to a cluster of basic amino acid residues and by electrostatic interactions between the negatively charged phosphate and these basic protein side chains. This binding site is in good agreement with earlier proposals [Banks et al., Nature (London) 279:773–777, 1979]. The phosphate oxygen atoms are hydrogen bonded to His‐62, Arg‐65, Arg‐122, and Arg‐170. The 2‐hydroxyl group, which defines the D‐isomer of 3PG, is hydrogen bonded to Asp‐23 and Asn‐25. The carboxyl group of 3‐PG points away from the N‐domain towards the C‐domain and is hydrogen bonded via a water molecule to main chain nitrogen atoms of helix‐14. The present structure of the 3‐PG‐bound pig muscle enzyme is compared with the structure of the substrate‐free horse enzyme. Major changes include an ordering of helix‐13 and a domain movement, which brings the N‐domain closer to the ATP‐binding C‐domain. This domain movement consists of a 7.7° rotation, which is less than previously estimated for the ternary complex. Local changes close to the 3‐PG bi

ISSN:0887-3585    

DOI:10.1002/prot.340120207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDiffuse scattering data have been collected on two crystal forms of lysozyme, tetragonal and triclinic, using synchrotron radiation. The observed diffraction patterns were simulated using an exact theory for simple model crystals which relates the diffuse scattering intensity distribution to the amplitudes and correlations of atomic movements. Although the mean square displacements in the tetragonal form are twice that in the triclinic crystal, the predominent component of atomic movement in both crystals is accounted for by short‐range coupled motions where displacement correlations decay exponentially as a function of atomic separation, with a relaxation distance of ≈ 6 Å. Lattice coupled movements with a correlation distance ≈ 50 Å account for only about 5–10% of the total atomic mean square displacements in the protein crystals. The results contradict various presumptions that the disorder in protein crystals can be modeled predominantly by elastic vibrations or rigid body

ISSN:0887-3585    

DOI:10.1002/prot.340120208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractComparison of the three‐dimensional structures of native endothiapepsin (EC 3.4.23.6) and 15 endothiapepsin oligopeptide inhibitor complexes defined at high resolution by X‐ray crystallography shows that endothiapepsin exists in two forms differing in the relative orientation of a domain comprising residues 190–302. There are relatively few interactions between the two parts of the enzyme; consequently, they can move as separate rigid bodies. A translational, librational, and screw analysis of the thermal parameters of endothiapepsin also supports and model in which the two parts can move relative to each other. In the comparison of different aspartic proteinases, the rms values are reduced by up to 47% when the two parts of the structure are superposed independently. This justifies description of the differences, including those between pepsinogen and pepsin (EC 3.4.34.1), as a rigid movement of one part relative to another although considerable distortions within the domains also occur. The consequence of the rigid body movement is a change in the shape of the active site cleft that is largest around the S3pocket. This is associated with a different position and conformation of the inhibitors that are bound to the two endothiapepsin forms. The relevance of these observations to a model of the hydrolysis by aspartic proteinases is briefly disc

ISSN:0887-3585    

DOI:10.1002/prot.340120209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline‐limited reaction that is unusually slow in the refolding of the wild‐type protein. The distribution of fast‐ and slow‐folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp‐59 is in close contact to Pro‐39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with thetrans→cisreisomerization of the Tyr‐38–Pro‐39 bond at the stage of a native‐like folding intermediate. The steric hindrance is abolished either by changing Trp‐59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild‐type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal thattrans→cisisomerization of Pro‐39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate ratelimiting steps by trappin

ISSN:0887-3585    

DOI:10.1002/prot.340120210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe gelatin‐binding region of fibronectin is easily isolated as a stable and functional 42‐kDa fragment (42‐kDa GBF) containing four type I “finger” modules and two type II “kringle‐like” modules arranged in the order I6–II1–II2–I7–I8–I9, where the numbers designate the order of these modules in each of the two polypeptide chains. Each module forms an independently folded domain stabilized by two disulfide bonds. Reduction of disulfides caused large changes in the intrinsic fluorescence and abolished the gelatin‐binding activity of 42‐kDa GBF and two nonoverlapping gelatin‐binding subfragments, 30‐kDa GBF (I6–II1–II2–I7) and 21‐kDa GBF (I8–I9). However, high yields of active material could be regenerated, without diluting the protein, by dialysis into GdmCl followed by slow overnight removal of GdmCl while maintaining the redox potential with a mixture of oxidized and reduced glutathione. Fluorescence spectroscopic analysis indicated that the tertiary structure and thermodynamic stability of the refolded fragments were similar to those of the originals. The refolded fragments were quantitatively indistinguishable from the originals with respect to their dissociation constants for binding to a fluorescent‐labeled collagen fragment. The results suggest that all or most of the cystines, a total of 24 in 42‐kDa GBF, are correctly paired in the refolded products and that the tertiary structure was completely recovered. The fact that the 30‐ and 21‐kDa fragments bind with a similar affinity proves the existence of at least two nonove

ISSN:0887-3585    

DOI:10.1002/prot.340120211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY