Protein Science
Công bố khoa học tiêu biểu
Sắp xếp:
Reassessing buried surface areas in protein–protein complexes Abstract The buried surface area (BSA), which measures the size of the interface in a protein–protein complex may differ from the accessible surface area (ASA) lost upon association (which we call DSA), if conformation changes take place. To evaluate the DSA, we measure the ASA of the interface atoms in the bound and unbound states of the components of 144 protein–protein complexes taken from the Protein–Protein Interaction Affinity Database of Kastritis et al . (2011). We observe differences exceeding 20%, and a systematic bias in the distribution. On average, the ASA calculated in the bound state of the components is 3.3% greater than in their unbound state, and the BSA, 7% greater than the DSA. The bias is observed even in complexes where the conformation changes are small. An examination of the bound and unbound structures points to a possible origin: local movements optimize contacts with the other component at the cost of internal contacts, and presumably also the binding free energy.
Protein Science - Tập 22 Số 10 - Trang 1453-1457 - 2013
Side‐chain conformational entropy at protein–protein interfaces Abstract Protein–protein interactions are the key to many biological processes. How proteins selectively and correctly associate with their required protein partner(s) is still unclear. Previous studies of this “protein‐docking problem” have found that shape complementarity is a major determinant of interaction, but the detailed balance of energy contributions to association remains unclear. This study estimates side‐chain conformational entropy (per unit solvent accessible area) for various protein surface regions, using a self‐consistent mean field calculation of rotamer probabilities. Interfacial surface regions were less flexible than the rest of the protein surface for calculations with monomers extracted from homodimer datasets in 21 of 25 cases, and in 8 of 9 for the large protomer from heterodimer datasets. In surface patch analysis, based on side‐chain conformational entropy, 68% of true interfaces were ranked top for the homodimer set and 66% for the large protomer/heterodimer set. The results indicate that addition of a side‐chain entropic term could significantly improve empirical calculations of protein–protein association.
Protein Science - Tập 11 Số 12 - Trang 2860-2870 - 2002
Prediction of residues in discontinuous B‐cell epitopes using protein 3D structures Abstract Discovery of discontinuous B‐cell epitopes is a major challenge in vaccine design. Previous epitope prediction methods have mostly been based on protein sequences and are not very effective. Here, we present DiscoTope, a novel method for discontinuous epitope prediction that uses protein three‐dimensional structural data. The method is based on amino acid statistics, spatial information, and surface accessibility in a compiled data set of discontinuous epitopes determined by X‐ray crystallography of antibody/antigen protein complexes. DiscoTope is the first method to focus explicitly on discontinuous epitopes. We show that the new structure‐based method has a better performance for predicting residues of discontinuous epitopes than methods based solely on sequence information, and that it can successfully predict epitope residues that have been identified by different techniques. DiscoTope detects 15.5% of residues located in discontinuous epitopes with a specificity of 95%. At this level of specificity, the conventional Parker hydrophilicity scale for predicting linear B‐cell epitopes identifies only 11.0% of residues located in discontinuous epitopes. Predictions by the DiscoTope method can guide experimental epitope mapping in both rational vaccine design and development of diagnostic tools, and may lead to more efficient epitope identification.
Protein Science - Tập 15 Số 11 - Trang 2558-2567 - 2006
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.
Protein Science - 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.
Protein Science - 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.
Protein Science - 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.
Protein Science - 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.
Protein Science - 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.
Protein Science - 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.
Protein Science - Tập 14 Số 8 - Trang 1975-1992 - 2005
Tổng số: 61
- 1
- 2
- 3
- 4
- 5
- 6
- 7