Protein Science
1469-896X
0961-8368
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Cơ quản chủ quản: WILEY , Wiley-Blackwell
Lĩnh vực:
BiochemistryMolecular BiologyMedicine (miscellaneous)
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Thông tin về tạp chí
Protein Science, the flagship journal of The Protein Society, serves an international forum for publishing original reports on all scientific aspects of protein molecules. The Journal publishes papers by leading scientists from all over the world that report on advances in the understanding of proteins in the broadest sense. Protein Science aims to unify this field by cutting across established disciplinary lines and focusing on “protein-centered” science. The Journal encompasses the structure, function, and biochemical significance of proteins, their role in molecular and cell biology, genetics, and evolution, and their regulation and mechanisms of action.
Các bài báo tiêu biểu
Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein Abstract The structure has been determined at 3.0 Å resolution of a complex of engineered monomeric Cro repressor with a seven‐base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half‐operator that is recognized by each subunit of the wild‐type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence‐independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48° further out of the major groove, while the turn region of the helix‐turn‐helix remains in contact with the DNA backbone. All of the direct base‐specific interactions seen in the wild‐type Cro‐operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein‐DNA interface than for half of the wild‐type Cro‐operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence‐nonspecific interaction of the wild‐type Cro dimer with DNA. In the sequence‐specific complex, the DNA is bent, the protein dimer undergoes a large hinge‐bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.
Tập 7 Số 7 - Trang 1485-1494 - 1998
Expansion of the genetic code: Site‐directed p‐fluoro‐phenylalanine incorporation in Escherichia coli Abstract Site‐directed incorporation of the amino acid analogue p ‐fluoro‐phenylalanine (p ‐F‐Phe) was achieved in Escherichia coli . A yeast suppressor tRNAPhe amber/phenylalanyl‐tRNA synthetase pair was expressed in an analogue‐resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64‐75% as p ‐F‐Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p ‐F‐Phe incorporation was 11‐21‐fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8‐12 mg/L of culture, corresponding to about two thirds of the expression level of the wild‐type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19 F‐NMR spectroscopy and other sample‐intensive methods. The use of a nonessential “21 st” tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
Tập 7 Số 2 - Trang 419-426 - 1998
Entropic barriers, transition states, funnels, and exponential protein folding kinetics: A simple model Abstract This paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time‐dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf‐course and funnel‐like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics.
Tập 9 Số 3 - Trang 452-465 - 2000
Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation Abstract A new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2 O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1 ) to very slow (k < 0.002 h−1 ). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c . For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.
Tập 2 Số 4 - Trang 522-531 - 1993
Natively unfolded proteins: A point where biology waits for physics Abstract The experimental material accumulated in the literature on the conformational behavior of intrinsically unstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity. Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premolten globules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poor solvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second group are essentially more compact, exhibiting some amount of residual secondary structure, although they are still less dense than native or molten globule proteins. An important feature of the intrinsically unstructured proteins is that they undergo disorder–order transition during or prior to their biological function. In this respect, the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globules, premolten globules, and random coils) and transitions between any two of the states, is discussed.
Tập 11 Số 4 - Trang 739-756 - 2002
Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide‐MHC without increasing apparent cross‐reactivity Abstract The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell‐surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide‐independent cross‐reactivity of native TCRs is generally avoided through cell‐mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line‐encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high‐affinity TCRs with therapeutic and diagnostic potential.
Tập 15 Số 4 - Trang 710-721 - 2006
The intrinsically disordered C‐terminal domain of the measles virus nucleoprotein interacts with the C‐terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded Abstract Measles virus is a negative‐sense, single‐stranded RNA virus within theMononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. Themeasles virus nucleoprotein consists of a structured N‐terminal domain, and of an intrinsically disordered C‐terminal domain, NTAIL (aa 401–525), which undergoes induced folding in the presence of the C‐terminal domain (XD, aa 459–507) of the viral phosphoprotein. With in NTAIL , an α‐helical molecular recognition element (α‐MoRE, aa 488–499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small‐angle X‐ray scattering, we have derived a low‐resolution structural model of the complex between XD and NTAIL , which shows that most of NTAIL remains disordered in the complex despite P‐induced folding within the α‐MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N‐terminal region of NTAIL , and of a bulky globular region, corresponding to XD and to the C terminus of NTAIL (aa 486–525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that NTAIL has an additional site (aa 517–525) involved in binding to XD but not in the unstructured‐to‐structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.
Tập 14 Số 8 - Trang 1975-1992 - 2005
The interaction of eIF4E with 4E‐BP1 is an induced fit to a completely disordered protein Abstract 4E binding protein 1 (4E‐BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4–10, 43–46, and 56–65 could not be assigned, primarily because of a high degree of 1 H and 15 N chemical shift overlap. Steady‐state {1 H}−15 N NOEs were measured for 45 residues in the assigned regions. Except for the two C‐terminal residues, the NOEs were between −0.77 and −1.14, indicating a high level of flexibility. Furthermore, the {1 H}−15 N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E‐BPI has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.
Tập 7 Số 7 - Trang 1639-1642 - 1998
Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two‐dimensional NMR Abstract In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein‐ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO‐D2 O mixtures was investigated by NMR methods. As in water, H‐D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid‐ and base‐catalyzed processes and negligible water catalysis. However, the base‐catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is ∼ 100‐fold reduced relative to that in D2 O. The solvent‐dependent decrease in base‐catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pK w seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent‐quenching method, melittin was exchanged in D2 O (pH 3.2, 12 °C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2 O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.
Tập 4 Số 4 - Trang 804-814 - 1995
Two‐promoter vector is highly efficient for overproduction of protein complexes Abstract The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two‐promoter vectors yielded four to nine times larger amounts of the complexes between BCL‐2 family proteins, BCL‐XL L
Tập 13 Số 6 - Trang 1698-1703 - 2004