A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids

Amino acids have distinct conformational preferences that influence the stabilities of protein secondary and tertiary structures. The relative thermodynamic stabilities of each of the 20 commonly occurring amino acids in the alpha-helical versus random coil states have been determined through the design of a peptide that forms a noncovalent alpha-helical dimer, which is in equilibrium with a randomly coiled monomeric state. The alpha helices in the dimer contain a single solvent-exposed site that is surrounded by small, neutral amino acid side chains. Each of the commonly occurring amino acids was substituted into this guest site, and the resulting equilibrium constants for the monomer-dimer equilibrium were determined to provide a list of free energy difference (delta delta G degree) values.     

Crystal structure of alpha 1: implications for protein design

X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.     

Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.     

The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins

A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.     

紫花苜蓿蛋白质二硫键异构酶mPDI的生物信息学分析与同源建模研究

利用紫花苜蓿蛋白质二硫键异构酶（mPDI）的mRNA（来自NCBI,登录号为Z11499．1）及氨基酸序列（来自UniProt KB／Swiss—Prot数据库及NCBI数据库,其登录号分别为1729828、CAA77575．1）,应用生物信息学软件预测了该蛋白质的理化性质、亲疏水性、信号肽、二级结构、卷曲螺旋结构、跨膜区域、糖基化位点、活性位点、亚细胞定位、功能结构域及高级结构。结果表明：紫花苜蓿mPDI蛋白质是一个整体疏水性蛋白,细胞定位为粗面内质网,含有512个氨基酸,理论等电点为4．98,分子量为57087．4Da,原子组成为C2597H3977N651O786S6摩尔消光系数在280nm处为37610,不稳定系数为40．12,脂肪系数为79．96,总平均亲水性为-0．357。该蛋白质由20种氨基酸组成,其中非极性R基团氨基酸占44．3％,极性R基团氨基酸占28．4％,酸性氨基酸占13．6％,碱性氨基酸占13．1％,Ip、Glu、Val、Ala含量最为丰富,Met和Cys含量最少。信号肽位于1～24号氨基酸。利用ProtScale软件的KyteandDoolittle算法对mPDI蛋白进行亲／疏水性预测,结果表明该蛋白质含有7个高疏水性区域,分别分布在4J0～50区域、95～105区域、120～130区域、190～200区域、285～295区域、340～350区域以及365～375区域。利用SignalP网络工具对mPDI蛋白进行信号肽的预测,神经网络法显示第24～25位点是最可能的剪切点,马可夫模型显示了同样的信号肽酶切位点。mPDI蛋白质二级结构中α-螺旋占26．37％（135AA）、无规则卷曲占53．32％（273AA）、延伸链占20．31％（104AA）;包含3个卷曲螺旋结构、3个糖基化位点（分别为143位的S、148位的T、461位的S）、2个硫氧还蛋白结构域（14～144位、357～485位）、2个硫氧还蛋白活性位点（54～72位、399～417位）、1个内质网靶向序列（509～512位）;Ramachandram结构检测表明此模型的三维结构符合立体化学能量规则。     

The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.     

Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.     

The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The approximately 3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.     

Scissors-grip model for DNA recognition by a family of leucine zipper proteins

C/EBP is a sequence-specific DNA binding protein that regulates gene expression in certain mammalian cells. The region of the C/EBP polypeptide required for specific recognition of DNA is related in amino acid sequence to other regulatory proteins, including the Fos and Jun transforming proteins. It has been proposed that these proteins bind DNA via a bipartite structural motif, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." An evaluation of the properties of conserved amino acids within the basic region of 11 deduced protein sequences, coupled with the observation that they are located at an invariant distance from the leucine zipper, has led to the formulation of a "scissors-grip" model for DNA binding. The architectural features of this model are well suited for interaction with directly abutted, dyadsymmetric DNA sequences. Data supportive of the model were obtained with chemical probes of protein: DNA complexes.     

Domain separation in the activation of glycogen phosphorylase a

The crystal structure of glycogen phosphorylase a complexed with its substrates, orthophosphate and maltopentaose, has been determined and refined at a resolution of 2.8 angstroms. With oligosaccaride bound at the glycogen storage site, the phosphate ion binds at the catalytic site and causes the regulatory and catalytic domains to separate with the loss of stabilizing interactions between them. Homotropic cooperativity between the active sites of the allosteric dimer results from rearrangements in isologous contacts between symmetry-related helices in the subunit interface. The conformational changes in the core of the interface are correlated with those observed on covalent activation by phosphorylation at Ser14 (phosphorylase b----a).